Download Genetic interactions between scribbler, Atrophin and

Genetics: Published Articles Ahead of Print, published on April 19, 2006 as 10.1534/genetics.105.055012
Genetic interactions between scribbler, Atrophin and groucho
uncover links in transcriptional repression
Amy Wehn and Gerard Campbell
Department of Biological Sciences
University of Pittsburgh
Pittsburgh, PA 15260
USA
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Wehn and Campbell: Gro, Sbb and Atro and repression
Running title: Gro, Sbb and Atro and repression
Key words: transcriptional repression, Groucho, Atrophin, Scribbler, Engrailed
Corresponding author:
Gerard Campbell, Ph.D.
Department of Biological Sciences
University of Pittsburgh
203 Life Sciences Annex
4259 Fifth Avenue
Pittsburgh, PA 15260
e-mail: [email protected]
Tel: (412) 624-6812
Fax: (412) 624-4759
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Wehn and Campbell: Gro, Sbb and Atro and repression
ABSTRACT
In eukaryotes, the ability of DNA binding proteins to act as transcriptional repressors often
requires that they recruit accessory proteins, known as corepressors, which provide the activity
responsible for silencing transcription. Several of these factors have been identified including
the Groucho (Gro) and Atrophin (Atro) proteins in Drosophila. Here we demonstrate strong
genetic interactions between gro and Atro and also with mutations in a third gene, scribbler
(sbb), which encodes a nuclear protein of unknown function. We show that mutations in Atro
and Sbb have similar phenotypes, including upregulation of the same genes in imaginal discs,
which suggests that Sbb cooperates with Atro to provide repressive activity. Comparison of gro
and Atro/sbb mutant phenotypes suggests they do not function together, but that they may
interact with the same transcription factors, including Engrailed and C15, to provide these
proteins with maximal repressive activity.
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Wehn and Campbell: Gro, Sbb and Atro and repression
INTRODUCTION
An essential feature of all eukaryotic cells is the ability to transcribe only a subset of the genes
present in their genomes. Whether a gene is transcribed or not is largely dependent upon the
presence and activity of specific regulatory transcription factors which bind in a sequencespecific manner to adjacent cis-regulatory elements. These regulatory transcription factors can
act either as activators and promote transcription or as repressors and inhibit it. To function as
repressors, these transcription factors often need to recruit accessory proteins known as
corepressors which provide the activity necessary to prevent transcription of a particular gene.
Several types of corepressor have been identified including Groucho (Gro), C-terminal
Binding Protein, Atrophin, N-CoR and SMRT (reviewed in GASTON and JAYARAMAN 2003).
These corepressors will prevent transcription when in proximity to a specific promoter or
activators; how they do this and how close they have to be to the promoter or activator binding
sites can vary among corepressors. However, in terms of mechanism, a common theme is the
ability to modify chromatin structure, often by recruiting histone deacetylase complexes;
deacetylation results in compaction of chromatin, which presumably hinders access of regulatory
and general transcription factors, thus shutting down transcription (DAVIE and DENT 2004).
Some transcription factors have been shown to recruit more than one corepressor, for
example, the Hairy and Brinker proteins from Drosophila have recruitment motifs for both CtBP
and Gro (HASSON et al. 2001; JIMENEZ et al. 1997; PAROUSH et al. 1994; PHIPPEN et al. 2000;
POORTINGA et al. 1998; ZHANG and LEVINE 1999). In theory, the ability to recruit more than one
corepressor can provide at least two possible advantages: first, quantitative, it may increase the
repressive activity of the transcription factor, and second, qualitative, one corepressor may be
more effective at repressing transcription of a specific gene than another. In reality it is often
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Wehn and Campbell: Gro, Sbb and Atro and repression
more difficult to understand exactly why a particular transcription factor recruits more than one
corepressor. For Brk, CtBP and Gro are largely redundant (HASSON et al. 2001; WINTER and
CAMPBELL 2004), while for Hairy there is evidence that it may selectively recruit each
corepressor for repression of specific targets (BIANCHI-FRIAS et al. 2004).
In this paper we present a genetic study that investigates the links between three different
proteins that appear to act as corepressors: Gro, Atro and another nuclear protein, Scribbler, the
specific function of which was previously unknown. New mutations in the genes encoding these
factors were identified in a genetic screen for enhancers of mutations in the aristaless (al) gene.
al encodes a transcription factor expressed at the presumptive tip of the leg and antenna, which
in combination with C15, another transcription factor expressed in the same cells, directs the
differentiation of the structures found there in adult appendages, the arista and tarsal claws
(CAMPBELL 2005; CAMPBELL and TOMLINSON 1998; KOJIMA et al. 2005; SCHNEITZ et al. 1993).
Gro belongs to the Gro/TLE family of related proteins, and is one of the most widely
studied corepressors (CHEN and COUREY 2000; FISHER and CAUDY 1998). It has been shown to
interact with many different transcriptional repressors including Hairy, Runt, Engrailed (En),
Even Skipped (Eve), Huckebein (Hkb) and Brk (ARONSON et al. 1997; FISHER et al. 1996;
GOLDSTEIN et al. 1999; HASSON et al. 2001; JIMENEZ et al. 1997; PAROUSH et al. 1994;
TOLKUNOVA et al. 1998; ZHANG et al. 2001) via a short interaction motif present in these
proteins, of which there are two general types: the WRPW tetrapeptide class and the eh1/GEH
octapeptide class (ARONSON et al. 1997; FISHER et al. 1996; GOLDSTEIN et al. 1999; JIMENEZ et
al. 1999; TOLKUNOVA et al. 1998). These proteins require Gro to have maximal repressive
activity, although most can repress by other mechanisms (ARONSON et al. 1997; FISHER et al.
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Wehn and Campbell: Gro, Sbb and Atro and repression
1996; GOLDSTEIN et al. 1999; HASSON et al. 2001; JIMENEZ et al. 1997; KOBAYASHI et al. 2001;
PAROUSH et al. 1994; ZHANG et al. 2001).
Drosophila Atro (aka Grunge), is a homolog of the Atrophin-1 protein of humans
(ERKNER et al. 2002; ZHANG et al. 2002), named because the expansion of a polyglutamine tract
is the cause of an inherited neuronal degenerative disease, dentatorubral-pallidoluysian atrophy
(KOIDE et al. 1994; NAGAFUCHI et al. 1994). Atro mutants in Drosophila have a range of
defects, including aberrant segmentation, neurogenesis and dorsoventral-patterning that are
associated with derepression of several genes (ERKNER et al. 2002; ZHANG et al. 2002). The role
of Atro as a corepressor was revealed by its direct interaction with the Eve and Hkb transcription
factors which appeared to be required for the normal repressive activities of these proteins.
Further, both Drosophila Atro and human Atrophin-1 can repress transcription when fused to a
heterologous DNA binding domain (ZHANG et al. 2002). Atro is a multifunctional protein and,
in addition to acting as a corepressor, it appears to be involved in two other processes. First, it
acts as a positive regulator of Hox gene expression and has been classed as a member of the
trithorax group of genes (KANKEL et al. 2004). Second, it has also been shown to function in the
cytoplasm where it interacts with the cytoplasmic domain of the atypical cadherin Fat, and is
required for establishment of planar cell polarity in the eye and wing (FANTO et al. 2003; ZHANG
et al. 2002).
The scribbler (sbb) gene (aka brakeless and master-of-thickveins) encodes a novel
nuclear protein of unknown function (FUNAKOSHI et al. 2001; RAO et al. 2000; SENTI et al. 2000;
YANG et al. 2000). sbb mutations were uncovered through three different phenotypes, first,
abnormal turning behavior in larvae (YANG et al. 2000), second, an axon targeting defect in the
eye where some photoreceptors project to the wrong location in the optic lobe (RAO et al. 2000;
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Wehn and Campbell: Gro, Sbb and Atro and repression
SENTI et al. 2000) and third, deregulation of expression of the thickveins (tkv) gene in the wing
imaginal disc (FUNAKOSHI et al. 2001). The sbb locus encodes two different isoforms, a short
one, SbbA, of 929 residues and a longer one, SbbB, of 2302 residues, which is a C-terminal
extension of SbbA (FUNAKOSHI et al. 2001; RAO et al. 2000; SENTI et al. 2000; YANG et al.
2000). Both isoforms are composed largely of low-complexity sequence with only a single
predicted functional domain, a zinc finger, in SbbB and none in SbbA. However, the C-terminus
of SbbA, extending into SbbB, has significant sequence homology to novel vertebrate proteins
(predicted from ESTs), indicating this is an important functional domain. Rescue experiments
demonstrated that the SbbA isoform is capable of rescuing most mutant phenotypes (FUNAKOSHI
et al. 2001; RAO et al. 2000; SENTI et al. 2000; YANG et al. 2000).
Mutations in sbb result in derepression of at least two genes, tkv in the wing and runt
(run) in the eye (FUNAKOSHI et al. 2001; KAMINKER et al. 2002). tkv encodes a BMP receptor
and is downregulated in the medial regions of the wing imaginal disc (TANIMOTO et al. 2000),
but not in sbb mutant cells where levels are equivalent to those laterally (FUNAKOSHI et al.
2001). Curiously the pattern of an enhancer trap in sbb mirrors that of tkv (FUNAKOSHI et al.
2001). However, elsewhere sbb appears to be uniformly expressed, including the eye (RAO et al.
2000; SENTI et al. 2000; YANG et al. 2000). Each ommatidium of the eye has eight
photoreceptors and the transcription factor Run is expressed only in photoreceptors R7 and R8.
This appears to be important for them to project their axons to the medulla in the optic lobe;
axons from R1-R6 project to the lamina instead (KAMINKER et al. 2002). Loss of sbb leads to
ectopic expression of run in R2 and R5 resulting in axons from all six outer photoreceptors, R1R6 projecting to the medulla (KAMINKER et al. 2002; RAO et al. 2000; SENTI et al. 2000). The
fact that Sbb is ubiquitously expressed in the eye but represses run in R2 and R5, and not R7 and
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Wehn and Campbell: Gro, Sbb and Atro and repression
R8, indicates either that its activity is modulated or that it could act as a corepressor for a
transcription factor expressed in R2/R5.
Here we show that Atro and sbb interact genetically and that mutations in each have very
similar phenotypes in some tissues, including derepression of the same genes, raising the
possibility that Sbb and Atro function together in a corepressor complex. In addition, these
genetic studies indicate that Atro/Sbb are required, at least in part, for the repressive activity of
two homeodomain transcription factors, C15 and Engrailed. We also show that gro interacts
genetically with sbb and Atro mutants, but that the phenotype of gro mutants is distinct from that
of Atro or sbb indicating Gro and Atro/Sbb function independently. The genetic interactions can,
however, be explained if both Gro and Atro/Sbb associate with the same transcription factors,
again including En and C15, and if both are required for the maximal activity of these factors.
MATERIALS AND METHODS
Fly strains: Flies carrying the following existing alleles or transgenes were used: alice, al130
(In(2L)al130), alush (not in Flybase; CAMPBELL 2005), hs-flp (P{hsFLP}22), FRT82B
(P{ry[+t7.2]=neoFRT}82B), Ubi-GFP (P{Ubi-GFP(S65T)nls}3R), M(2)201, M(3)95A
(RpS3Plac92), sbb6 (previously, mtv6), Atro35 (Gug35), hs-GFP (on 2R is Avic\GFPhs.T:Ivir\HA, on 3L
is Avic\GFPhs.T:Hsap\MYC), groE48, arm-lacZ (Ecol\lacZarm.PV), M(2)IK (not in Flybase; ZECCA and
STRUHL 2002), M(3)i55 (RpS174), enE (Df(2L)enE), FRTG13 (P{w[+mW.hs]=FRT(w[hs])}G13),
ovoD (P{w[+mC]=ovoD1-18}2R), FRT42D (P{ry[+t7.2]=neoFRT}42D), y+ (y+t7.7), FRT2A
(P{w[+mW.hs]=FRT(w[hs])}2A), tkv-lacZ (tkv04415), dpp-lacZ (dpp10638), hs-CD2 (P{hsp70CD2.J}), mwh1, y1. Unless indicated otherwise in parentheses, all genotypes are as denoted in
Flybase (http://flybase.bio.indiana.edu), where more information on each can be found.
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Wehn and Campbell: Gro, Sbb and Atro and repression
al enhancer screen: This screen has been described previously and enhancers were
characterized as mutations in the Egfr and C15 genes (CAMPBELL 2005).
Clonal analysis in adults and imaginal discs: Homozygous mutant clones in imaginal
discs and adults were generated in imaginal discs by hs-flp/FRT-induced mitotic recombination
(XU and RUBIN 1993). Clones were generated in the second or early third instar of larvae with
the following genotypes:
y hs-flp; FRT42D sbb6/ FRT42D hs-GFP M(2)IK y+
y hs-flp; FRT42D enE/ FRT42D hs-GFP M(2)IK y+
hs-flp; dpp-lacZ FRT42D enE/ FRT42D hs-GFP M(2)IK y+
y hs-flp; Atro35 FRT2A/ M(3)i55 y+ FRT2A
hs-flp; mwh1 Atro35 FRT2A/ M(3)i55 hs-GFP FRT2A
hs-flp; tkv-lacZ/+; Atro35 FRT2A/ M(3)i55 hs-GFP FRT2A
hs-flp; FRT82B groE48/ FRT82B Ubi-GFP M(3)95A
y hs-flp; dpp-lacZ/+; FRT82B groE48/ FRT82B Ubi-GFP M(3)95A
y hs-flp; tkv-lacZ/ +; FRT82B groE48/ FRT82B hs-CD2 M(3)95A
hs-flp; FRT42D sbb6/ FRT42D arm-lacZ M(2)60E; FRT82B groE48/ FRT82B Ubi-GFP M(3)95A
Clones were identified in adults by loss of y+ and in discs by the loss of GFP or CD2 expression.
Germline clones: embryos mutant for both maternal and zygotic sbb were generated by
the dominant autosomal germline clone technique (CHOU and PERRIMON 1992) using females
with the following genotype:
hs-flp; FRTG13 sbb6/ FRTG13 ovoD
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Wehn and Campbell: Gro, Sbb and Atro and repression
Clones were induced in late third instar larvae and the resulting adults were crossed to sbb6/Cyo
males. It was not possible to identify which progeny received the sbb6 or the Cyo chromosome,
but as almost exactly 50% died (with a specific defect) it was assumed these were homozygous
for sbb6 and that the wild-type sbb gene on Cyo could rescue the loss of maternal contribution in
the remaining 50%.
Immunostaining and cuticle preps: Dissection and staining of imaginal discs was
carried out by standard techniques. The following antibodies were used: anti-Al (rat; 1:1,000)
(CAMPBELL et al. 1993); anti-B (rabbit, 1:5) (HIGASHIJIMA et al. 1992); anti-ßgal (rabbit,
Cappell, 1:2000 and mouse, Promega, 1:200), anti-Ci (2A1, rat, 1:1) (SLUSARSKI et al. 1995);
anti-En (ascites, 1:500) (PATEL et al. 1989); anti-Run (1:500) (KOSMAN et al. 1998). Secondary
antibodies were from Jackson immunochemicals (Cy5 conjugates, at 1:200) and Molecular
Probes (Alexa 488 and Alexa 568 conjugates at 1:500). Tissue from adult flies was mounted in
GMM (LAWRENCE and JOHNSTON 1986). First instar cuticle preps were prepared by an initial
clearing in actic acid:glycerol (4:1; 30 mins at 60oC, 24 hr at room temp) and then mounting in a
3:1 mixture of CMCP-10 mounting medium (Polysciences):lactic acid.
RESULTS
A genetic screen for enhancers of al uncovers mutations in gro, Atro and sbb
The aristaless (al) gene encodes a homeodomain transcription factor that is expressed in the
center of the leg and antennal imaginal discs of Drosophila (CAMPBELL et al. 1993; SCHNEITZ et
al. 1993) (Fig. 1A). This region gives rise to the distal-most structures of the respective
appendage, the claw organ in the adult leg and the arista in the antenna; in al mutants both of
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Wehn and Campbell: Gro, Sbb and Atro and repression
these structures are absent or reduced (Fig. 2A,B) (CAMPBELL and TOMLINSON 1998).
Previously, we have described a genetic screen that identified genes operating upstream, Egfr,
and in parallel, C15, to al (CAMPBELL 2005). EGF-receptor signaling is required for activation
of al (CAMPBELL 2002; GALINDO et al. 2002), while C15 encodes another homeodomain protein
which is expressed in the same cells as Al. Al and C15 function together to regulate gene
expression in the center of the leg and antennal discs (CAMPBELL 2005; KOJIMA et al. 2005).
This screen also uncovered six additional enhancers 4, 13, 15, 17, 37 and 73 (Fig. 2C-F),
which corresponded to three different complementation groups, 4/17, 13/37, 15/73, and not to
Egfr or C15. Mapping by recombination and/or deficiencies and subsequent complementation
testing against known genes in potential locations of these enhancers revealed that 4/17 is allelic
to scribbler (sbb), 13/37 to groucho (gro) and 15/73 to Atrophin (Atro). Null alleles and
deficiencies covering sbb (Df(2L)PC4) and gro (Df(3R)Espl22) also act as enhancers. In
contrast, Atro35, a null allele (ERKNER et al. 2002) is a weaker enhancer than 15 or 73 suggesting
these may have dominant negative activity. The six enhancers have been renamed, sbbEal4,
sbbEal17, groEal13, groEal37, AtroEal15 and AtroEal73.
sbb and Atro mutations have similar effects on patterning of the aristae and claws
Next we compared the phenotype of al mutations to that of gro, Atro, and sbb mutations in a
wild-type al background, first in adults and then in imaginal discs. Strong al mutants survive to
adult in which both the arista (Fig. 2B) and the claw organ (consisting of a pair of claws, a pair
of pulvilli and an empodium) are absent (CAMPBELL and TOMLINSON 1998). Of the six
enhancers, direct analysis of homozygotes was only possible in sbbEal17 because the other five
enhancers were either embryonic or larval lethal as homozygotes. Similarly, previously
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Wehn and Campbell: Gro, Sbb and Atro and repression
characterized null alleles of Atro and gro, including Atro35 and groE58, are embryonic lethal,
while sbb nulls, such as sbb6, survive embryogenesis but die during the larval phase (ERKNER et
al. 2002; FUNAKOSHI et al. 2001; KANKEL et al. 2004; RAO et al. 2000; SENTI et al. 2000; YANG
et al. 2000; ZHANG et al. 2002). sbbEal17 is, thus, weaker than a null allele and sequencing
revealed a premature stop in this mutant truncating the long isoform (SbbB) at residue 1620 and
eliminating 682 residues at the C-terminus, the sequence of the short isoform (SbbA) being
normal (Fig. 3A).
sbbEal17 homozytoges actually die as pharate adults, and this permitted examination of
their antennae and legs, revealing their aristae to be wild-type (not shown) but that their legs
occasionally had a severely reduced tarsal claw (Fig. 2I, about 7% had at least one claw
affected); the other structures of the claw organ, the pulvilli and empodium, were unaffected.
sbbEal17/6 flies also survive to pharate adults in which defective claws are found at a higher
frequency (24%). The effect of complete loss of Sbb was examined in sbb6 homozygous clones
and was found to result in severe reduction in the size, particularly the width, of claws comprised
of mutant tissue (Fig. 2K). Patterning of sbb6 mutant antennae was also disrupted so that the
aristae were reduced in length and fatter (Fig. 2H); in addition, some sense organs on the third
antennal segment, including the sensilla trichodea, were absent or lacking setae (not shown).
The claw phenotype is similar to that found in the hypomorphic al130 mutant (CAMPBELL and
TOMLINSON 1998) suggesting Al activity is reduced in the absence of Sbb. However, the arista
phenotype is distinct from that in al hypomorphs, which have aristae that resemble those in wildtype, but are shorter and thinner (STERN and BRIDGES 1926).
Atro mutant clones (both AtroEal15 and Atro35) at the tip of the antenna and leg produced
phenotypes very similar to that of sbb6 clones, i.e shorter, fatter aristae and reduced claws
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resembling bristles (Fig. 2G, J). In contrast, as already reported, gro mutant clones (both gro13
and groE48) are not recovered in adults (HEITZLER et al. 1996).
gro, Atro and sbb mutants show partial derepression of B
Al, in combination with C15, represses expression of genes such as Bar (B) in the center of the
leg and antennal discs; in wild-type discs B is absent from the center and is expressed in a ring
surrounding Al (Fig. 1A); in al mutants, however, B is expressed throughout the center
(CAMPBELL 2005; CAMPBELL et al. 1993; KOJIMA et al. 2000; KOJIMA et al. 2005). Given that
Gro and Atro are corepressors, an obvious possibility was that one or both interact with Al
and/or with C15 to facilitate repression of B. Previous studies indicated that Al alone does not
act as a transcriptional repressor and, in fact, can activate expression of B in the absence of C15
(CAMPBELL 2005). Consistent with this, no Gro interaction motifs could be identified in Al.
C15, however, possesses an eh1 class Gro interaction motif first identified in the Engrailed (En)
homeodomain protein (Fig. 3B) (JIMENEZ et al. 1999; TOLKUNOVA et al. 1998); this motif is
conserved among vertebrate homologs of C15 (Fig. 3Bii). More direct studies are required to
determine if Atro physically interacts with C15 because, although Atro has been shown to
interact directly with other transcription factors, the specific sequence required has not yet been
characterized (ZHANG et al. 2002).
To determine if Gro and/or Atro are required to provide repressive activity to C15/Al, B
expression was examined in leg discs containing gro and Atro mutant clones. This revealed that
there was some derepression of B in both gro and Atro mutant cells in the center (Fig. 1B, C), but
that this was much less dramatic than with al or C15 mutant clones (CAMPBELL 2005). More
specifically, the B expression domain expands into the center in gro and Atro mutant cells, but
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Wehn and Campbell: Gro, Sbb and Atro and repression
there is always a small region in the center where B is still repressed (Fig. 1B, C). sbb mutant
clones also have minor effects on B expression, although this appeared to be less consistent and
severe than with gro or Atro clones (Fig. 1D). Thus, loss of Gro or Atro does result in partial
derepression of B but this is much weaker than loss of Al or C15 demonstrating that C15/Al can
still repress B in the absence of either Gro or Atro corepressors, although not as effectively. One
possibility is that Gro and Atro act partially redundantly in this respect so that in the absence of
both, C15/Al will not be able to repress B. Testing this by generating gro Atro double mutant
clones is technically difficult as each is on a different arm of the third chromosome. However,
further experiments, described below, indicate that Atro requires Sbb to provide repressor
activity to transcription factors; this would also account for the very similar phenotypes of Atro
and sbb clones at the tip of the leg and antenna. Consequently, the phenotype of sbb gro double
mutant clones was investigated (technically not that difficult as each is on separate chromosome)
and it was found that B was still repressed in the very center (Fig. 1E). This indicates that Atro
may function as a corepressor in the absence of Sbb or that Al/C15 can use other mechanisms to
repress B in addition to Atro/Sbb and Gro.
Atro and sbb mutations have similar phenotypes outside of the tip of the leg and antenna
As already described, sbb and Atro mutant clones produce similar phenotypes at the tip of the leg
and antenna; this is also true in other locations in the adult. First, patterning of the dorsal region
of the head around the ocelli is disrupted in both sbb and Atro mutants. In wild-type adults this
region, known as the ocellar triangle, consists of 3 well spaced ocelli, 6-8 small interocellar
bristles and two large ocellar bristles (Fig. 4A). In sbbEal17 homozygotes and sbbEal17/6 adults
there is a compression of this region, with fusion of ocelli and loss or mispositioning of the
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Wehn and Campbell: Gro, Sbb and Atro and repression
interocellar and ocellar bristles (Fig. 4B, C). sbb6 or Atro35 mutant clones result in the loss of
almost all of these elements, with a single ocellus often being the only structure remaining (Fig.
4D, E). Second, it has been reported already that Atro mutant clones can generate fusion of
segments in the leg (ERKNER et al. 2002; KANKEL et al. 2004) (Fig. 2L) and this is also true for
sbb mutant clones (not shown). This is also seen quite dramatically in about 5% of the legs from
sbbEal17 homozygotes (Fig. 2M; curiously this phenotype is stronger in sbbEal17 homozygotes than
in sbbEal17/6 adults). The most dramatic fusions are actually seen between adjacent prothoracic
legs; this is also true for Atro clones (Fig. 2L). Third, both Atro and sbb clones produce a similar
phenotype at the margin of the wing. In proximal anterior regions, the triple row of wild-type
wings includes a large, wide central bristle; in both Atro and sbb mutant clones these bristles are
thinner, and in Atro but not sbb clones they are longer (Fig. 2N, O). In the posterior of wild-type
wings, the hairs at the margin do not have a socket, but sbb and Atro mutant hairs do possess a
small socket and are slightly longer than wild-type margin hairs. This is indicative of a partial
transformation to anterior where all the bristles at the margin possess a socket.
It should be noted, however, that sbb and Atro mutant clones do not always produce
similar phenotypes. For example, Atro mutant clones have been shown to disrupt planar polarity
(FANTO et al. 2003; ZHANG et al. 2002); sbb mutant clones have no effect on bristle or hair
orientation in the wing (not shown).
Atro and sbb mutations have identical effects on tkv and run expression in imaginal discs
The similarity between the phenotype of sbb and Atro mutant clones in adults suggested that Sbb
and Atro may function together in a corepressor complex. This predicted that any gene
derepressed in cells lacking Atro would be derepressed in cells lacking Sbb, and vice versa. This
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Wehn and Campbell: Gro, Sbb and Atro and repression
was tested in imaginal discs: previous studies have revealed that loss of Sbb results in
upregulation of tkv expression in the anterior compartment of the wing and misexpression of run
in the eye (FUNAKOSHI et al. 2001; KAMINKER et al. 2002). It was found that Atro mutant clones
show an identical phenotype (Fig. 5A,B, D, E), supporting the argument that Sbb is required for
Atro to function as a corepressor.
sbb mutant embryos have a similar but weaker phenotype than Atro mutants
A significant difference between Atro and sbb mutants is that null Atro mutants are embryonic
lethal while null sbb mutants survive embryogenesis and die as larvae (ERKNER et al. 2002;
FUNAKOSHI et al. 2001; KANKEL et al. 2004; RAO et al. 2000; SENTI et al. 2000; YANG et al.
2000; ZHANG et al. 2002). Although Atro mutants are lethal, the severity of this lethal phenotype
is made significantly worse by removing maternal contribution with germline clones (ERKNER et
al. 2002; KANKEL et al. 2004; ZHANG et al. 2002). Consequently, we tested whether sbb mutant
embryos may be rescued by maternal contribution and found that loss of both maternal and
zygotic sbb resulted in embryonic lethality (embryos lacking only maternal contribution survive
embryogenesis); and although there was some variability, these embryos had a distinct segmental
phenotype consisting of the deletion of three or more abdominal segments (Fig. 6A, B).
Analysis of En expression, which marks the posterior of each segment, revealed that most mutant
embryos had defects in alternate segments, primarily A4, 6 and 8, corresponding to evennumbered En stripes in parasegments 10, 12 and 14 (Fig. 6C, D). This is similar to the
phenotype of Atro mutant embryos which also have defects in even-numbered En stripes
(KANKEL et al. 2004). However, this Atro mutant phenotype is associated with embryos that are
lacking maternal but not zygotic contribution; embryos that lack both have a very severe
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Wehn and Campbell: Gro, Sbb and Atro and repression
phenotype and in many cases appear as if they have not been fertilized (ERKNER et al. 2002;
KANKEL et al. 2004; ZHANG et al. 2002). Consequently, although the sbb embryonic phenotype
is very similar to that of Atro mutants, it is not as severe.
Dominant genetic interactions between Atro, sbb and gro
Although the initial complementation tests revealed that the Atro mutations picked up as
enhancers of al were not alleles of sbb because they were on different chromosomes, it was
noticed that sbb/+; Atro/+ transheterozygous adults had weak phenotypes similar to homozygous
single mutants of Atro and sbb. This was most profound in the dorsal head where patterning of
the ocellar triangle was invariably disrupted; this was manifested in fusion of ocelli,
mispositioning of ocellar bristles and loss of interocellar bristles (Fig. 4F). This interaction was
observed between Atro and sbb alleles uncovered in the al enhancer screen, and also between
extant alleles, sbb6 and Atro35; and so was not associated with preexisting mutations in the stocks
used in this screen. It proved possible to quantitate this phenotype by simply counting the
number of interocellar bristles in adults of different genotypes (Fig. 7). Wild-type adults have an
average of 7.3 (±1.1) interocellar bristles (Fig. 7A) and, surprisingly, adults heterozygous for
either sbb or Atro mutations (including the likely null alleles, Atro35 and sbb6) have a significant
reduction in this number indicating that each has a very weak dominant phenotype, presumably
due to haploinsufficiency, at least for the null alleles (Fig. 7B). This number is significantly
reduced further in sbb/+; Atro/+ transheterozygotes (Fig. 7C).
Further analysis revealed that gro mutations also interacted dominantly with sbb and Atro
mutations (Fig. 4G), so that gro heterozygous adults also have a significant reduction in the
17
Wehn and Campbell: Gro, Sbb and Atro and repression
number of ocellar bristles (Fig. 7B) and this is reduced further in both sbb/+; gro/+ and Atro +/ +
gro transheterozygotes (Fig. 7D).
sbb/Atro mutants have distinct phenotypes from that of gro mutants
The dominant genetic interactions between sbb and Atro can be explained if Sbb and Atro
proteins function together; this can also account for the very similar phenotypes of sbb and Atro
homozygous clones in imaginal discs and adults described above. This posed the question of
whether the dominant genetic interactions between gro and Atro or sbb can also be explained if
Atro, Sbb and Gro function together as a corepressor complex. This was tested by comparing the
phenotype of gro mutant clones in imaginal discs to those of Atro and sbb. As already described,
both Atro and sbb clones have an identical effect on tkv and run expression in the wing and eye
disc, respectively. In contrast, it was found that loss of gro had no effect on tkv expression in the
wing (Fig. 5C), while gro mutant clones in the eye had a different phenotype to that of Atro and
sbb, resulting primarily in loss of run expression rather than ectopic expression (Fig. 5F, G).
During the course of this study, however, it was found that run was ectopically expressed in gro
mutant clones located in the antennal disc. In wild-type antennal discs Run is expressed in a
small number of cells in three clusters in the presumptive third antennal segment; in gro mutant
clones the number of Run expressing cells in this disc increases dramatically (Fig. 5H, J). In
contrast, Run expression in antennal discs containing Atro or sbb mutant clones is
indistinguishable from wild-type (Fig. 5I, and data not shown). Thus, there are clear differences
between the effects of loss of Atro/Sbb and loss of Gro on gene expression so that, for example,
Atro-dependent repression of tkv in the wing is independent of Gro, while Gro-dependent
repression of run in the antenna is independent of Atro/Sbb.
18
Wehn and Campbell: Gro, Sbb and Atro and repression
Dominant genetic interactions between Gro/Atro/sbb and en
What is the basis of the dominant genetic interactions between gro and Atro or sbb? One
possibility is that Gro and Atro/Sbb interact with the same transcription factor that represses gene
expression in the presumptive ocellar triangle, and that Gro and Atro/Sbb are both required for
maximal activity of this factor. This is the same explanation used for Al/C15 to explain why
gro, Atro and sbb all act as enhancers of al. A candidate for this transcription factor required for
patterning the ocellar triangle was Engrailed (En) which has been reported to be expressed in this
region (ROYET and FINKELSTEIN 1996) and is known to have a Gro interaction motif, the original
eh1 motif (JIMENEZ et al. 1997; JIMENEZ et al. 1999; TOLKUNOVA et al. 1998). In addition, the
posterior wing margin phenotype of Atro and sbb mutant clones is consistent with a weak
posterior to anterior transformation that could be explained by a reduction in En activity, En
being required to establish posterior cell fates (GARCIA-BELLIDO and SANTAMARIA 1972).
This possibility was tested first by determining if there were any dominant genetic
interactions between en and gro, Atro or sbb (the en allele used in these studies was Df(2L)enE
which takes out both en and its partially redundant partner, invected (inv) (TABATA et al. 1995)).
Unlike gro, Atro and sbb mutations, enE is not haploinsufficient and heterozygotes have a normal
number of interocellar bristles (Fig. 7A). However, enE transheterozygotes with some, but not
all, gro, Atro and sbb alleles show a significant reduction in the number of these bristles (Fig.
4H, I, 7E). This possibility was tested further by examining the phenotype of enE clones in the
head and it was found that these resulted in almost complete loss of the ocellar triangle with a
single ocellus remaining (Fig. 4J).
19
Wehn and Campbell: Gro, Sbb and Atro and repression
De-repression of one but not other En-target genes in gro, Atro or sbb mutant cells
The genetic interactions between en and gro, Atro and sbb predicted that loss of En in the
presumptive ocellar triangle in the eye imaginal disc might result in de-repression of some of the
same genes in gro and/or Atro/sbb mutant clones. A direct test of this is not possible at present
because there are no known targets of En in the presumptive ocellar triangle. Consequently, we
decided to study this question in the wing imaginal disc where several targets of En have been
identified, including decapentaplegic (dpp) and cubitus interruptus (ci); En is expressed in the
posterior compartment of the wing, while ci is expressed throughout the anterior and dpp in a
stripe immediately anterior to the compartment boundary (Fig. 8A, F, I) (BROWER 1986; EATON
and KORNBERG 1990; MASUCCI et al. 1990; ORENIC et al. 1990; RAFTERY et al. 1991; SANICOLA
et al. 1995; SCHWARTZ et al. 1995; TABATA et al. 1995; ZECCA et al. 1995). Consistent with
previous studies, we find that both ci and dpp are ectopically expressed in en mutant clones in
the posterior (Fig. 8F, I). We have also identified another En target in the wing: al; unlike the
leg, where it is expressed in both anterior and posterior compartments, in the wing al is restricted
to the anterior (Fig. 8A). As for ci and dpp, clonal analysis reveals that this restriction is Endependent (Fig. 8B).
Next we tested whether repression of ci, dpp and al by En was dependent upon Gro, Atro
or Sbb. Both ci and dpp expression is unaffected in gro, Atro or sbb single mutant clones in the
posterior (Fig. 8G, H, J, K and not shown), indicating that En can repress these targets
completely in the absence of one of these factors. In contrast, there is some ectopic expression
of al in gro, Atro or sbb mutant clones (Fig. 8C-E). However, this derepression is only partial so
that the ectopic al expression is not as extensive as with loss of en. These results indicate that En
can repress ci and dpp completely even in the absence of either Gro, Atro or Sbb, but that
20
Wehn and Campbell: Gro, Sbb and Atro and repression
repression of al is partially dependent upon Gro, Atro or Sbb. Similar to Al/C15-dependent
repression of B in the leg, one possibility was that Gro and Atro/Sbb may act redundantly (for ci
and dpp) or partially redundantly (for al) in En-dependent repression. As already mentioned,
generating gro Atro double mutant clones is technically difficult, but we did analyze sbb gro
double mutant clones and found that there was very weak derepression of ci in the posterior in
cells lacking both Sbb and Gro (Fig. 8L, M). This shows that Gro and Sbb are at least partially
redundant, but that En is still very active in the absence of both. As for Al/C15, we can conclude
either that En uses mechanisms to repress that do not involve Gro and Atro/Sbb, or that Atro has
some activity in the absence of Sbb. Resolution of this will await analysis of Atro gro double
mutant clones.
DISCUSSION
Sbb is required for Atro activity
Previous studies demonstrated that Atro acts as a corepressor in Drosophila; the most convincing
of these being the demonstration that fusion of Atro to a heterologous DNA binding domain
confers repressive activity to the chimera (ZHANG et al. 2002). Atro has been shown to interact
directly with two transcription factors, Even-Skipped (Eve) and Huckebein and the repressive
ability of Eve is compromised in Atro mutants (ZHANG et al. 2002), probably accounting for the
loss of en expression in even number parasegments in Atro mutant embryos (KANKEL et al.
2004).
Our studies here are consistent with Atro acting as a corepressor as we show that several
genes, including run, tkv, al and B, are completely or partially derepressed in Atro mutant clones
in imaginal discs (Figs. 1C, 5B, E, 8D), suggesting that transcriptional repressors required to
21
Wehn and Campbell: Gro, Sbb and Atro and repression
silence these genes recruit Atro. Atro-dependent repression of B in the center of the leg disc is
very likely due to interaction with the transcription factor C15, which is expressed in the center
of the leg and is required for repression of B (CAMPBELL 2005; KOJIMA et al. 2005). Similarly,
Atro-dependent repression of al in the posterior of the wing is very likely due to interaction with
En, which is expressed in the posterior and required to exclude al from this compartment (Fig.
8A, B). At present it is unclear which transcription factors recruit Atro to repress run in the eye
or tkv in the wing, although a strong candidate for run would be the Rough homeodomain protein
(TOMLINSON et al. 1988) which is expressed in the same cells, R2 and R5, where run is
ectopically expressed in Atro mutant clones. Whether Atro can, in fact, bind directly to C15, En
and possibly Rough, needs to be tested biochemically, as previous studies with Eve and Hkb
(ZHANG et al. 2002) did not identify a possible interaction motif for Atro nor do sequence
comparisons between C15, En and Eve and Hkb suggest a common motif.
The sbb gene encodes a nuclear protein with unknown function (FUNAKOSHI et al. 2001;
RAO et al. 2000; SENTI et al. 2000; YANG et al. 2000). sbb mutations have many different
phenotypes affecting multiple tissues. Here we show that sbb and Atro interact very strongly
genetically (Fig. 4F, 7) and that many of the phenotypes of sbb mutants are very similar to those
of Atro mutants, including derepession of run, tkv, al and B in imaginal discs (Fig. 1C, D, 5B,E)
(FUNAKOSHI et al. 2001; KAMINKER et al. 2002). Thus, Atro is unable to silence these genes in
the absence of Sbb, suggesting that it is either required for Atro activity, to recruit Atro to
transcription factors or possibly to assist binding of these factors to DNA. As these transcription
factors appear to function normally in some respects in the absence of Sbb (or Atro), it appears
more likely that Sbb and Atro function together in a corepressor complex.
22
Wehn and Campbell: Gro, Sbb and Atro and repression
One problem with the proposal that Atro activity is dependent upon Sbb is that the
phenotypes of Atro and sbb mutants are not identical. For example, embryos lacking both
maternal and zygotic Atro have a very severe, almost uncharacterizable phenotype (ERKNER et
al. 2002; KANKEL et al. 2004; ZHANG et al. 2002), while embryos lacking both maternal and
zygotic Sbb have a much less severe phenotype, characterized by a reduced number of
abdominal segments (Fig. 6B), that is similar to that of embryos lacking only maternal Atro.
This could be explained if Atro is partially active in the absence of Sbb, or if it is dependent upon
Sbb for repression of some genes but not others. Alternatively, the difference between Atro and
sbb mutant phenotypes could be related to Atro having functions other than that of a corepressor.
It is has been implicated in positive regulation of Hox gene expression (KANKEL et al. 2004), and
it also functions in the cytoplasm to control planar cell polarity (FANTO et al. 2003; ZHANG et al.
2002). Our analysis of sbb mutants does not reveal any potential involvement of Hox gene
expression or planar cell polarity and consequently, if Sbb is only required for Atro to act as a
corepressor, then it is not surprising that Atro and sbb mutant phenotypes are not identical.
Further experiments are required to determine the nature of the Atro-dependence on Sbb for
transcriptional repression and how direct any interactions might be.
Some transcription factors may recruit both Gro and Atro/Sbb for maximal activity
We originally uncovered mutations in sbb and Atro in a genetic screen for enhancers of al. As
already mentioned, it is likely that they act as enhancers because they are utilized by the C15
transcription factor to repress genes such as B; C15 is expressed in the same cells as Al and it is
thought that they bind together to regulate gene expression (CAMPBELL 2005; KOJIMA et al.
23
Wehn and Campbell: Gro, Sbb and Atro and repression
2005). We have also uncovered strong genetic interactions between sbb, Atro and en mutations,
which can be explained if En also recruits Atro/Sbb.
Curiously, our genetic studies also revealed strong interactions between gro and sbb and
Atro. This could be explained if Gro was also required for Atro activity, i.e. that all three
proteins may form a corepressor complex. However, this appears to be unlikely because, in
contrast to the similar phenotypes of sbb and Atro mutants, there are several distinct differences
among the phenotype of gro mutants and those of sbb and Atro mutants. For example,
repression of tkv in the anterior of the wing is dependent on both Sbb and Atro but not Gro (Fig.
5B, C) (FUNAKOSHI et al. 2001), while repression of run in the antennal disc is dependent upon
Gro but not Atro or Sbb (Fig. 5H, I; data not shown). This suggests that there is a specific
transcription factor that recruits Atro/Sbb to repress tkv in the wing and another transcription
factor that recruits Gro to repress run in the antenna. The identity of these transcription factors
remains to be uncovered.
In some cases gro mutants do have a similar phenotype to that of Atro and sbb; this
includes partial derepression of al expression in the posterior of the wing and B in the center of
the leg (Figs. 1B, C, 8C, D). This can be explained if C15 (expressed in the center of the leg)
and En (expressed in the posterior of the wing) recruit both Gro and Atro/Sbb and each imparts
some but not all the repressive activity to these transcription factors. Consistent with this, both
C15 and En possess eh1-type Gro-interaction motifs (Fig. 3) (JIMENEZ et al. 1997; JIMENEZ et al.
1999; TOLKUNOVA et al. 1998) and previous studies have revealed that En can repress in the
absence of Gro (TOLKUNOVA et al. 1998). As already mentioned, further biochemical studies are
required to determine if C15 and En can indeed recruit Atro.
24
Wehn and Campbell: Gro, Sbb and Atro and repression
At present it is unclear whether Atro and Gro provide all the repressive activity to C15
and En; this will await the generation of Atro gro double mutant clones. We have, however,
analyzed sbb gro double mutant clones and these reveal that some targets of C15 and En are still
at least partially repressed, although En activity appears to be somewhat compromised following
the simultaneous loss of Sbb and Gro, in comparison to loss of one alone (Fig. 1E, 8M). Either
Atro has some activity in the absence of Sbb, or C15 and En can use mechanisms other than
recruitment of Gro and Atro to repress transcription. Many transcription factors have been
shown to have the ability to repress by several mechanisms, for example, although Brk recruits
both CtBP and Gro, it can repress some genes in the absence of both, using additional repression
domains (WINTER and CAMPBELL 2004).
Why do C15 and En need to recruit both Gro and Atro? En can repress some genes
completely in the absence of either Gro or Atro, for example ci and dpp in the wing (Fig. 8 G, H,
J, K). However, for repression of al, the activity of En is clearly reduced in the absence of either
indicating that it needs to recruit both to completely repress this gene (Fig. 8C, D). This would
suggest a quantitative explanation, i.e. En recruits both Gro and Atro to increase its activity,
rather than to allow it to repress specific genes repressed more efficiently by one or the other.
This is consistent with the suggestion that both corepressors function via a similar mechanism:
both Gro and a vertebrate homolog of Atro have been shown to recruit a histone deacetylase
(CHEN et al. 1999; ZOLTEWICZ et al. 2004). The recruitment of both may decrease histone
acetylation to a level that cannot be achieved with either alone.
25
Wehn and Campbell: Gro, Sbb and Atro and repression
ACKNOWLEDGEMENTS
We thank A. Bodnar for technical assistance and to T. Welch for some preliminary studies. The
screen for enhancers of al was begun in Andrew Tomlinson’s lab and GC thanks him for
support. We are indebted to Pascal Heitzler for the alush allele. We thank the following people
for materials used in this study: C. Alonso, S. Cohen, B. Dickson, S. Kerridge, R. Holmgren, T.
Kojima, K. Matthews, K. Cook and the Bloomington Stock Center, Y. Rao, J. Reinitz, K. Senti,
M. Sokolowski, G. Struhl, T. Tabata, M. Zecca, We thank, D. Chapman and B. Stronach for
comments on the manuscript. This work was supported by grants from the National Institute of
Health (GM60368) and March of Dimes (#1-FY02-176) to GC.
REFERENCES
ARONSON, B. D., A. L. FISHER, K. BLECHMAN, M. CAUDY and J. P. GERGEN, 1997 Grouchodependent and -independent repression activities of Runt domain proteins. Mol Cell Biol
17: 5581-5587.
BIANCHI-FRIAS, D., A. ORIAN, J. J. DELROW, J. VAZQUEZ, A. E. ROSALES-NIEVES et al., 2004
Hairy transcriptional repression targets and cofactor recruitment in Drosophila. PLoS
Biol 2: E178.
BLAIR, S. S., 1992 Engrailed expression in the anterior lineage compartment of the developing
wing blade of Drosophila. Development 115: 21--33.
BROWER, D. L., 1986 Engrailed gene expression in Drosophila imaginal discs. Embo J 5: 26492656.
CAMPBELL, G., 2002 Distalization of the Drosophila leg by graded EGF-receptor activity. Nature
418: 781-785.
CAMPBELL, G., 2005 Regulation of gene expression in the distal region of the Drosophila leg by
the Hox11 homolog, C15. Dev Biol 278: 607-618.
CAMPBELL, G., and A. TOMLINSON, 1998 The roles of the homeobox genes aristaless and Distalless in patterning the legs and wings of Drosophila. Development 125: 4483-4493.
CAMPBELL, G., T. WEAVER and A. TOMLINSON, 1993 Axis specification in the developing
Drosophila appendage: the role of wingless, decapentaplegic, and the homeobox gene
aristaless. Cell 74: 1113-1123.
CHEN, G., and A. J. COUREY, 2000 Groucho/TLE family proteins and transcriptional repression.
Gene 249: 1-16.
26
Wehn and Campbell: Gro, Sbb and Atro and repression
CHEN, G., J. FERNANDEZ, S. MISCHE and A. J. COUREY, 1999 A functional interaction between
the histone deacetylase Rpd3 and the corepressor groucho in Drosophila development.
Genes Dev 13: 2218-2230.
CHOU, T. B., and N. PERRIMON, 1992 Use of a yeast site-specific recombinase to produce female
germline chimeras in Drosophila. Genetics 131: 643-653.
DAVIE, J. K., and S. Y. DENT, 2004 Histone modifications in corepressor functions. Curr Top
Dev Biol 59: 145-163.
EATON, S., and T. B. KORNBERG, 1990 Repression of ci-D in posterior compartments of
Drosophila by engrailed. Genes Dev 4: 1068-1077.
ERKNER, A., A. ROURE, B. CHARROUX, M. DELAAGE, N. HOLWAY et al., 2002 Grunge, related to
human Atrophin-like proteins, has multiple functions in Drosophila development.
Development 129: 1119-1129.
FANTO, M., L. CLAYTON, J. MEREDITH, K. HARDIMAN, B. CHARROUX et al., 2003 The tumorsuppressor and cell adhesion molecule Fat controls planar polarity via physical
interactions with Atrophin, a transcriptional co-repressor. Development 130: 763-774.
FISHER, A. L., and M. CAUDY, 1998 Groucho proteins: transcriptional corepressors for specific
subsets of DNA-binding transcription factors in vertebrates and invertebrates. Genes Dev
12: 1931-1940.
FISHER, A. L., S. OHSAKO and M. CAUDY, 1996 The WRPW motif of the hairy-related basic
helix-loop-helix repressor proteins acts as a 4-amino-acid transcription repression and
protein-protein interaction domain. Mol Cell Biol 16: 2670-2677.
FUNAKOSHI, Y., M. MINAMI and T. TABATA, 2001 mtv shapes the activity gradient of the Dpp
morphogen through regulation of thickveins. Development 128: 67-74.
GALINDO, M. I., S. A. BISHOP, S. GREIG and J. P. COUSO, 2002 Leg patterning driven by
proximal-distal interactions and EGFR signaling. Science 297: 256-259.
GARCIA-BELLIDO, A., and P. SANTAMARIA, 1972 Developmental analysis of the wing disc in the
mutant engrailed of Drosophila melanogaster. Genetics 72: 87-104.
GASTON, K., and P. S. JAYARAMAN, 2003 Transcriptional repression in eukaryotes: repressors
and repression mechanisms. Cell Mol Life Sci 60: 721-741.
GOLDSTEIN, R. E., G. JIMENEZ, O. COOK, D. GUR and Z. PAROUSH, 1999 Huckebein repressor
activity in Drosophila terminal patterning is mediated by Groucho. Development 126:
3747-3755.
HASSON, P., B. MULLER, K. BASLER and Z. PAROUSH, 2001 Brinker requires two corepressors
for maximal and versatile repression in Dpp signalling. Embo J 20: 5725-5736.
HEITZLER, P., M. BOUROUIS, L. RUEL, C. CARTERET and P. SIMPSON, 1996 Genes of the
Enhancer of split and achaete-scute complexes are required for a regulatory loop between
Notch and Delta during lateral signalling in Drosophila. Development 122: 161-171.
HIGASHIJIMA, S., T. KOJIMA, T. MICHIUE, S. ISHIMARU, Y. EMORI et al., 1992 Dual Bar homeo
box genes of Drosophila required in two photoreceptor cells, R1 and R6, and primary
pigment cells for normal eye development. Genes Dev 6: 50-60.
JIMENEZ, G., Z. PAROUSH and D. ISH-HOROWICZ, 1997 Groucho acts as a corepressor for a
subset of negative regulators, including Hairy and Engrailed. Genes Dev 11: 3072-3082.
JIMENEZ, G., C. P. VERRIJZER and D. ISH-HOROWICZ, 1999 A conserved motif in goosecoid
mediates groucho-dependent repression in Drosophila embryos. Mol Cell Biol 19: 20802087.
27
Wehn and Campbell: Gro, Sbb and Atro and repression
KAMINKER, J. S., J. CANON, I. SALECKER and U. BANERJEE, 2002 Control of photoreceptor axon
target choice by transcriptional repression of Runt. Nat Neurosci 5: 746-750.
KANKEL, M. W., D. M. DUNCAN and I. DUNCAN, 2004 A screen for genes that interact with the
Drosophila pair-rule segmentation gene fushi tarazu. Genetics 168: 161-180.
KOBAYASHI, M., R. E. GOLDSTEIN, M. FUJIOKA, Z. PAROUSH and J. B. JAYNES, 2001 Groucho
augments the repression of multiple Even skipped target genes in establishing
parasegment boundaries. Development 128: 1805-1815.
KOIDE, R., T. IKEUCHI, O. ONODERA, H. TANAKA, S. IGARASHI et al., 1994 Unstable expansion
of CAG repeat in hereditary dentatorubral-pallidoluysian atrophy (DRPLA). Nat Genet 6:
9-13.
KOJIMA, T., M. SATO and K. SAIGO, 2000 Formation and specification of distal leg segments in
Drosophila by dual Bar homeobox genes, BarH1 and BarH2. Development 127: 769-778.
KOJIMA, T., T. TSUJI and K. SAIGO, 2005 A concerted action of a paired-type homeobox gene,
aristaless, and a homolog of Hox11/tlx homeobox gene, clawless, is essential for the
distal tip development of the Drosophila leg. Dev Biol 279: 434-445.
KOSMAN, D., S. SMALL and J. REINITZ, 1998 Rapid preparation of a panel of polyclonal
antibodies to Drosophila segmentation proteins. Dev Genes Evol 208: 290-294.
LAWRENCE, P. A., and P. JOHNSTON, 1986 Methods of marking cells., pp. 229-242 in
Drosophila: a Practical Approach, edited by D. B. ROBERTS. IRL Press, Oxford.
MASUCCI, J. D., R. J. MILTENBERGER and F. M. HOFFMANN, 1990 Pattern-specific expression of
the Drosophila decapentaplegic gene in imaginal disks is regulated by 3' cis-regulatory
elements. Genes Dev 4: 2011-2023.
NAGAFUCHI, S., H. YANAGISAWA, E. OHSAKI, T. SHIRAYAMA, K. TADOKORO et al., 1994
Structure and expression of the gene responsible for the triplet repeat disorder,
dentatorubral and pallidoluysian atrophy (DRPLA). Nat Genet 8: 177-182.
ORENIC, T. V., D. C. SLUSARSKI, K. L. KROLL and R. A. HOLMGREN, 1990 Cloning and
characterization of the segment polarity gene cubitus interruptus Dominant of
Drosophila. Genes Dev. 4: 1053--1067.
PAROUSH, Z., R. L. FINLEY, JR., T. KIDD, S. M. WAINWRIGHT, P. W. INGHAM et al., 1994
Groucho is required for Drosophila neurogenesis, segmentation, and sex determination
and interacts directly with hairy-related bHLH proteins. Cell 79: 805-815.
PATEL, N. H., E. MARTIN-BLANCO, K. G. COLEMAN, S. J. POOLE, M. C. ELLIS et al., 1989
Expression of engrailed proteins in arthropods, annelids, and chordates. Cell 58: 955-968.
PHIPPEN, T. M., A. L. SWEIGART, M. MONIWA, A. KRUMM, J. R. DAVIE et al., 2000 Drosophila
C-terminal binding protein functions as a context-dependent transcriptional co-factor and
interferes with both mad and groucho transcriptional repression. J Biol Chem 275:
37628-37637.
POORTINGA, G., M. WATANABE and S. M. PARKHURST, 1998 Drosophila CtBP: a Hairyinteracting protein required for embryonic segmentation and hairy-mediated
transcriptional repression. Embo J 17: 2067-2078.
RAFTERY, L. A., M. SANICOLA, R. K. BLACKMAN and W. M. GELBART, 1991 The relationship of
decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes
mark the anterior-posterior compartment boundary? Development 113: 27-33.
RAO, Y., P. PANG, W. RUAN, D. GUNNING and S. L. ZIPURSKY, 2000 brakeless is required for
photoreceptor growth-cone targeting in Drosophila. Proc Natl Acad Sci U S A 97: 59665971.
28
Wehn and Campbell: Gro, Sbb and Atro and repression
ROYET, J., and R. FINKELSTEIN, 1996 hedgehog, wingless and orthodenticle specify adult head
development in Drosophila. Development 122: 1849-1858.
SANICOLA, M., J. J. SEKELSKY, S. ELSON and W. M. GELBART, 1995 Drawing a stripe in
Drosophila imaginal disks: Negative regulation of decapentaplegic and patched
expression by engrailed. Genetics 139: 745--756.
SCHNEITZ, K., P. SPIELMANN and M. NOLL, 1993 Molecular genetics of aristaless, a prd-type
homeo box gene involved in the morphogenesis of proximal and distal pattern elements
in a subset of appendages in Drosophila [published erratum appears in Genes Dev 1993
May;7(5):911]. Genes Dev 7: 114-129.
SCHWARTZ, C., J. LOCKE, C. NISHIDA and T. B. KORNBERG, 1995 Analysis of cubitus interruptus
regulation in Drosophila embryos and imaginal disks. Development 121: 1625--1635.
SENTI, K., K. KELEMAN, F. EISENHABER and B. J. DICKSON, 2000 brakeless is required for
lamina targeting of R1-R6 axons in the Drosophila visual system. Development 127:
2291-2301.
SLUSARSKI, D. C., C. K. MOTZNY and R. HOLMGREN, 1995 Mutations that alter the timing and
pattern of cubitus interruptus gene expression in Drosophila melanogaster. Genetics 139:
229--240.
STERN, C., and C. V. BRIDGES, 1926 The mutants of the extreme left end of the second
chromosome of Drosophila melanogaster. Genetics 11: 503-530.
TABATA, T., C. SCHWARTZ, E. GUSTAVSON, Z. ALI and T. B. KORNBERG, 1995 Creating a
Drosophila wing de novo, the role of engrailed, and the compartment border hypothesis.
Development 121: 3359--3369.
TANIMOTO, H., S. ITOH, P. TEN DIJKE and T. TABATA, 2000 Hedgehog creates a gradient of DPP
activity in Drosophila wing imaginal discs. Mol Cell 5: 59-71.
TOLKUNOVA, E. N., M. FUJIOKA, M. KOBAYASHI, D. DEKA and J. B. JAYNES, 1998 Two distinct
types of repression domain in engrailed: one interacts with the groucho corepressor and is
preferentially active on integrated target genes. Mol Cell Biol 18: 2804-2814.
TOMLINSON, A., B. E. KIMMEL and G. M. RUBIN, 1988 rough, a Drosophila homeobox gene
required in photoreceptors R2 and R5 for inductive interactions in the developing eye.
Cell 55: 771-784.
WINTER, S. E., and G. CAMPBELL, 2004 Repression of Dpp targets in the Drosophila wing by
Brinker. Development 131: 6071-6081.
XU, T., and G. M. RUBIN, 1993 Analysis of genetic mosaics in developing and adult Drosophila
tissues. Development 117: 1223-1237.
YANG, P., S. A. SHAVER, A. J. HILLIKER and M. B. SOKOLOWSKI, 2000 Abnormal turning
behavior in Drosophila larvae. Identification and molecular analysis of scribbler (sbb).
Genetics 155: 1161-1174.
ZECCA, M., K. BASLER and G. STRUHL, 1995 Sequential organizing activities of engrailed,
hedgehog and decapentaplegic in the Drosophila wing. Development 121: 2265-2278.
ZECCA, M., and G. STRUHL, 2002 Subdivision of the Drosophila wing imaginal disc by EGFRmediated signaling. Development 129: 1357-1368.
ZHANG, H., and M. LEVINE, 1999 Groucho and dCtBP mediate separate pathways of
transcriptional repression in the Drosophila embryo. Proc Natl Acad Sci U S A 96: 535540.
ZHANG, H., M. LEVINE and H. L. ASHE, 2001 Brinker is a sequence-specific transcriptional
repressor in the Drosophila embryo. Genes Dev 15: 261-266.
29
Wehn and Campbell: Gro, Sbb and Atro and repression
ZHANG, S., L. XU, J. LEE and T. XU, 2002 Drosophila atrophin homolog functions as a
transcriptional corepressor in multiple developmental processes. Cell 108: 45-56.
ZOLTEWICZ, J. S., N. J. STEWART, R. LEUNG and A. S. PETERSON, 2004 Atrophin 2 recruits
histone deacetylase and is required for the function of multiple signaling centers during
mouse embryogenesis. Development 131: 3-14.
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FIGURE LEGENDS
Figure 1. Bar (B) expression (red) in leg discs containing gro, Atro and sbb mutant clones
(clones are identified by the loss of a ubiquitous marker; green, GFP in Bii, Cii, Dii, Eii and blue,
ß-gal in Eii, and, thus, appear black). (A) In wild-type leg discs, Al (green) is expressed in a
central domain and is surrounded by a ring of B expression. The Al domain is divided (white
line) into two halves corresponding to the anterior and posterior compartments, which are almost
identical in size. (Aii) Magnification of the central region showing complete absence of B in the
center apart from two spots of expression corresponding to the tarsal claws. (B) B is partially
derepressed in gro mutant clones. A clone bisects the center, its position and straight margin
indicates that it comprises all of the anterior compartment in the center (the non-B expressing
cells; asterisk); this is smaller than the wild-type posterior, indicating that B expression has
expanded into the center, also evidenced by the B domain being wider than in wild-type discs.
In the very center (asterisk, magnified in iii), B is still almost completely repressed in gro mutant
cells, although there is actually some very weak B expression in some cells here (Biii). (C) Atro
clones are similar to gro mutant clones. This disc also contains a mutant clone that bisects the
center, here the posterior (non-B expressing cells, asterisk) is comprised of Atro mutant cells and
is smaller than the wild-type anterior due to expansion of B into the center. (Ciii) Also, although
B is almost completely repressed in Atro mutant cells in the very center, there is some weak
derepression, that is actually more apparent than in the gro mutant cells in (Biii). (D) There is
occasional expansion of the B domain into the center (arrow) in sbb mutant clones. (E) This disc
contains clones of both sbb and gro; in the very center there is a clone of cells mutant for both
sbb and gro (arrowhead). B is not deregulated in this clone.
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Wehn and Campbell: Gro, Sbb and Atro and repression
Figure 2. al enhancer screen and the phenotype of sbb and Atro mutant legs and antenna. (A-H)
Adult antennae. (A) Wild-type with the arista labeled (ar). (B) alice mutant in which the arista is
almost completely absent. (C) Weaker al130/ush mutant which has an almost full-length arista.
(D-F) Also al130/ush mutants, but now heterozygous for AtroEal15 (D), sbbEal17(E), and groEal13 (F).
These mutations dominantly enhance the arista phenotype of al130/ush mutants. (G) Antenna
comprised almost entirely of AtroEal15 mutant clones; the arista is fatter and shorter with reduced
lateral branches. (H) Antenna comprised almost entirely of sbb6 mutant clones; the arista is very
similar to that in (G). (I-K) Tips of adult legs. (I) sbbEal17 homozygote, which has one normal
claw (i) and associated pulvillus (p) and a defective claw (ii), but the associated pulvillus is
normal. (J) Leg containing AtroEal15 clones; (i) this claw is completely mutant and is much
narrower than the adjacent claw (ii) which is comprised of wild-type cells. (K) Claw from a sbb6
mutant clone; this is also narrower. (L) Adult prothoracic legs comprised almost entirely (apart
from the claws) of AtroEal15 clones. The legs have fused and segmentation of the medial and
proximal regions is almost absent. (M) Prothoracic legs from sbbEal17 homozygote; these are
also fused and have defective segmentation. A partial duplication is also present. (N-Q) The
margin from adult wings containing Atro35 or sbb6 mutant clones. (N-O) Anterior, proximal
margin which normally develops the triple row of bristles including a large peg-like bristle; wildtype bristles are indicated (TR). These large bristles are narrower in Atro and sbb mutant tissue,
and longer in Atro- tissue. (P-Q) Posterior margin which normally develops posterior row
bristles that do not have a socket (PR in P). These bristles have a socket and are longer in Atro
(P) and sbb (Q) mutant tissue.
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Wehn and Campbell: Gro, Sbb and Atro and repression
Figure 3. Sbb and C15 proteins. (A) sbb encodes two protein isoforms, SbbA and SbbB, which
are made up largely of low complexity sequence, except for three regions that show similarity to
predicted vertebrate proteins (indicated as 1-3) (FUNAKOSHI et al. 2001; RAO et al. 2000; SENTI
et al. 2000; YANG et al. 2000)). The mutation in sbbEal17 introduces a stop codon after residue
1620 truncating the SbbB isoform, but leaving the SbbA isoform unchanged. (Bi) The C15
protein contains a eh1 Gro interaction motif situated near the N-terminus. (Bii) Vertebrate
homologs of C15 also possess this motif at the N-terminus.
Figure 4. Defects in patterning of the ocellar triangle in sbb, Atro, gro, and en mutant tissue and
in transheterozygotes. (A) In wild-type adults this region of the dorsal head comprises 3 ocelli
(arrowheads), 2 large ocellar bristles (b) and 6-8 smaller interocellar bristles (i). (B, C) In
sbbEal17 homozygotes and sbbEal17/6 adults this region is compressed resulting in fusion of ocelli,
loss of interocellar bristles and loss and mispositioning of the ocellar bristles. (D, E) Heads
containing sbb6 and Atro35 mutant clones result in more severe patterning defects in which
almost all of this region is lost, arrows mark a single ocellus (note, it is not possible to identify
mutant tissue in the region affected because the marker used, y, only marks the bristles and no
mutant interocellar or ocellar bristles are ever identified). (F, G) Transheterozygotes between
sbbEal17, AtroEa;15 or groEal13 also have defects in patterning in this region, but these are less
extreme. (H, I) Transheterozygotes between enL11 and Atro and gro also have defective ocellar
triangles, here the number of interocellar bristles is reduced. (J) enL11 mutant clones result in
almost complete loss of the ocellar triangle, here a single enlarged ocellus remains.
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Wehn and Campbell: Gro, Sbb and Atro and repression
Figure 5. Comparison of Atro and gro mutant clones in imaginal discs. Clones appear black
and are identified by loss of a ubiquitous marker (red, GFP or CD2). (A) In wild-type wing
discs, tkv is expressed at relatively high levels posterior to the compartment boundary (arrow);
but anterior to the boundary levels are very low and then increase laterally. (B) Wing disc
containing Atro mutant clones (black); tkv (green) is upregulated in clones in the anterior
(arrowheads). (C) Wing disc containing gro mutant clones; these have no effect on tkv
expression (green). (D, E) Eye disc containing Atro mutant clones, E is a magnification of the
box in D. In wild-type tissue (red), Run (green) is expressed in a single photoreceptor per
ommatidium (yellow arrowhead, this is R8; in older ommatidia it is also expressed in R7 which
is in a different plane; photoreceptors are identified by Elav staining, blue). In mutant tissue
there are three Run positive photoreceptors per ommatidium (white arrowhead). (F, G) Eye disc
containing gro mutant clones, G is a magnification of the box in F. Mutant tissue (white
arrowhead) is largely devoid of Run expressing cells except at the margin, although mutant cells
do express the neural specific Elav marker. (H-J) Antennal discs. (H) In wild-type antennal
discs Run is expressed in a small number of cells in three clusters in the third segment. (I) This
pattern appears almost identical in this disc containing Atro mutant clones. (J) gro clones,
however, result in a dramatic increase in the number of cells expressing Run.
Figure 6. sbb germ line clones. (A-B) Cuticle preps of first instar larvae. (A) Wild-type with
eight abdominal segments (1-8). (B) sbb mutant; the number of abdominal segments is reduced
to four. (C) Wild-type germband extended embryo stained for En expression. (D) sbb maternal
and zygotic mutant embryo stained for En, there are defects in some of the stripes in the
abdomen (asterisks).
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Wehn and Campbell: Gro, Sbb and Atro and repression
Figure 7. Genetic interactions between sbb, Atro, gro, en revealed by a reduction in the number
of interocellar bristles in adult transheterozygotes. The genotype of the adults is given below the
histogram. (A) wild-type; (B) heterozygotes for only a single mutation; (C-E)
transheterozygotes, i.e. the adults also had a wild-type copy of mutations 1 and 2. To determine
if there was a significant change in the number of interocellar bristles, Student t-tests were
performed comparing the number of bristles to that of both mutations in single heterozygotes (in
B). Almost all of the genotypes showed a significant reduction in the number of bristles. (Error
bars indicate the standard error of the mean.)
Figure 8 Deregulation of gene expression in en, gro, Atro and sbb mutant clones in wing discs.
Clones are identified by the loss of a ubiquitous marker (red or blue). (A – E) Al expression
(green). (A) Wild-type disc. En (red) is expressed in the posterior (to the left in all discs), Al is
expressed in the anterior in a fan-shaped pattern (the slight overlap with En is probably explained
by the anterior expansion of En in the late third instar (BLAIR 1992)). (B) Disc composed almost
entirely of en clones showing expansion of the Al expression domain into the posterior (arrow)
forming a mirror-image of that in the anterior. (C) Disc almost completely composed of gro
clones showing expansion of the Al domain into the posterior, but the domain here is smaller
than in the anterior (expression in the anterior is also expanded slightly). (D, E) Al is also
ectopically expressed in posterior Atro and sbb clones (arrows), but this is much more restricted
compared to expression in the anterior. (F–H) Ci (green) is normally expressed only in the
anterior but is ectopically expressed in posterior en clones (F, arrows). However, expression is
normal in discs composed almost entirely of gro (G) or Atro (H) clones. (I-K) dpp is normally
expressed only in a stripe immediately anterior to the compartment boundary. As for Ci, dpp is
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Wehn and Campbell: Gro, Sbb and Atro and repression
misexpressed in posterior en clones (I), but loss of gro (J) or Atro (K) has no effect on expression
in the posterior. (L, M) Disc containing both gro (loss of blue) and sbb (loss of red) clones, cells
mutant for both appear as black. M is a magnification of the box in L. In the posterior, Ci is
very weakly ectopically expressed in cells mutant for both gro and sbb (arrow).
36