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First published online 31 January 2007
doi: 10.1242/dev.02796
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Department of Biology, Duke University, Durham, NC 27708, USA.
* Author for correspondence (e-mail: bejsovec{at}duke.edu)
Accepted 2 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: SoxN, Wg, Wnt, Drosophila, Embryo, Signal transduction
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Logan and Nusse, 2004). In
vertebrate embryos, different Wnt proteins promote a variety of cell-fate
decisions, including the patterning of the limbs, brain and other organs. In
Drosophila, a single Wnt molecule, encoded by the wingless
(wg) gene, directs a remarkably similar set of patterning events.
Excess Wnt pathway activity in vertebrate tissues is associated with a variety
of cancers, particularly colorectal tumors (reviewed in
Bienz and Clevers, 2000;
Polakis, 2000). Although
excess Wg activity in flies does not create tumors, it does produce profound
tissue alterations that are easily detected. All of the cell surface and
intracellular Wnt pathway components are highly conserved between vertebrates
and Drosophila, and many were first identified in Drosophila
through mutational disruptions of patterning (reviewed in
Bejsovec, 2006).
At the end of Drosophila embryogenesis, epidermal cells secrete a
highly patterned cuticle layer. The ventral surface displays segmental
denticle belts, which are `tractor tread' arrays of hooked elements
interspersed with `naked' cuticle. wg loss-of-function mutant embryos
secrete a uniform lawn of denticles, lacking the naked cuticle that should
separate the belts (Fig. 1C,
Fig. 2A). High levels of Wg
signaling convert all of the ventral epidermis to the naked cuticle cell fate
(Noordermeer et al., 1992;
Hays et al., 1997). Mutations
in downstream components that positively activate the pathway show a
wg-like phenotype, whereas mutations in negative regulators show an
excess-naked-cuticle phenotype (reviewed in
Dierick and Bejsovec,
1999).
Epistasis experiments with these mutations have shown that the pathway
hinges on the regulation of Armadillo (Arm), which is the fly beta-catenin
homolog (reviewed in Bejsovec,
2000; Peifer and Polakis,
2000; Jones and Bejsovec,
2003). In the absence of Wg signaling, Arm levels are kept low by
a set of proteins known collectively as the destruction complex. These
proteins, which include the Axin and Apc scaffolding molecules and the
serinethreonine kinase Shaggy (also known as Zeste-white3, Zw3; GSK3ß in
vertebrates), target Arm for destruction via ubiquitylation. When Wg binds to
its receptor complex, which consists of Arrow (LRP5/6 in vertebrates) and
Frizzled, this inactivates the destruction complex and allows Arm to
accumulate. In the simplest view, Arm accumulation drives its interaction with
Tcf, an HMG-box transcription factor, in the nucleus. Tcf can bind DNA in the
absence of Arm and represses Wg target gene expression in conjunction with
Groucho (Gro), a transcriptional co-repressor
(Cavallo et al., 1998;
Roose et al., 1998). When Arm
binds to Tcf, it displaces Gro and recruits other proteins to form a
transcriptional activation complex that promotes Wg target gene expression
(Brunner et al., 1997;
van de Wetering et al., 1997;
Kramps et al., 2002;
Daniels and Weis, 2005).
The balance between repression and activation properties of the Tcf complex
is crucial for Wnt target gene regulation. Recent work suggests that some
negative regulators, such as Apc and GSK3ß, may act in the nucleus in
regulatory complexes at the promoters of Wnt target genes
(Sierra et al., 2006), and
that these complexes act at least in part through chromatin remodeling.
However, the mechanism that switches Tcf from repressor to activator is still
unclear. To identify new molecules that regulate Wg/Wnt pathway activity, we
have performed genetic screens in Drosophila for mutations that
suppress weak wg loss-of-function phenotypes. Here, we describe a
strong suppressor mutation that partially rescues both hypomorphic and null
mutant alleles of wg. This mutation is allelic with previously
isolated mutations in SoxNeuro (SoxN), a gene that is
required for the specification of neural progenitors in the embryonic central
nervous system (Buescher et al.,
2002; Overton et al.,
2002).
Sox proteins, like Tcf proteins, contain HMG domains, which bind DNA. SoxN
is most closely related to vertebrate Class B Sox family members - Sox1, Sox2
and Sox3 (Cremazy et al.,
2000; Cremazy et al.,
2001) - which control cell-fate decisions in developmental
processes ranging from sex determination to chondrogenesis (reviewed in
Kamachi et al., 2000;
Wilson and Koopman, 2002). In
addition, Xenopus XSox3, as well as XSox17
and XSox17ß
(Zorn et al., 1999;
Sinner et al., 2004), and the
mouse Sox9 protein (Akiyama et al.,
2004) interfere with Wnt signaling by physically interacting with
beta-catenin through their C-termini. However, other work suggests that the
interaction with beta-catenin is not sufficient to explain the in vivo
Wnt-modulating function. Zhang et al.
(Zhang et al., 2003) found
that the DNA-binding domain, rather than the beta-catenin-binding region, is
crucial for the influence of XSox3 proteins on Wnt target expression. Our data
support the idea that Sox proteins act as true repressors in vivo. We show
that SoxN strongly represses Wg/Wnt-mediated target gene transcription, and we
find no evidence for an interaction of SoxN with Arm. Instead, we detect a
strong genetic interaction with Tcf, suggesting that SoxN is involved in the
delicate balance between the repressor and activator functions of Tcf.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
wgCX4 is an RNA-null allele
(Baker, 1987). Existing
SoxN mutations (SoxNGA1192, SoxNC463
and SoxNC2139) and UAS-SoxN flies were kindly
provided by G. Tear and S. Russell, respectively
(Seeger et al., 1993;
Overton et al., 2002).
UAS-Tcf and UAS-TcfDN (also known as
N) are as described by van de Wetering et al.
(van de Wetering et al.,
1997). Ubiquitous embryonic expression of UAS transgenes
was achieved with either the E22C-Gal4 or the arm-Gal4
driver lines (both from the Bloomington Stock Center). dpp-blink-Gal4
drives UAS transgene expression in a stripe along the anteroposterior
boundary of each imaginal disc (Johnston
and Schubiger, 1996). Epistasis experiments were performed with
the strong arm4 allele (armYD35) and
were verified with the weaker arm8
(armH8.6) allele
(Peifer and Wieschaus, 1990)
and the strong Tcf2 allele
(van de Wetering et al.,
1997). The gro deficiency groBX22
(Cavallo et al., 1998) is also
known as Df(3R)Espl22.
Most Gal4-UAS experiments were conducted at 28°C to allow
maximal activity of the Gal4 protein. Experiments involving the
dpp-Gal4 driver were performed at 25°C because of increased
lethality at the higher temperature. Hatching efficiencies and cuticle
preparation were performed as described by Jones and Bejsovec
(Jones and Bejsovec, 2005),
except that all hatched larvae were also mounted for scoring. Because balancer
chromosomes can introduce non-specific cuticle pattern defects, epistasis
crosses were performed without marked balancers.
Isolation and characterization of the NC14 mutation
Details of the EMS mutagenesis are described by Jones and Bejsovec
(Jones and Bejsovec, 2005).
The NC14 mutation was mapped by standard meiotic recombination with
adult-visible mutations. The Bloomington Deficiency Kit stocks
Df(2L)N22-14 and Df(2L)N22-2 uncover the genetic interval
containing the mutation, which was refined further by its complementation of
Df(2L)Exel7039 and failure to complement Df(2L)Exel7040.
Male site-specific recombination (Chen
et al., 1998) was used to pinpoint the mutation with respect to
molecularly characterized P-element insertions. A marked NC14 mutant
chromosome crossed into a 2-3 background was placed in trans
to P-bearing chromosomes. Males were crossed back to flies with the same
marked NC14 chromosome to score viable recombinant progeny. For
candidate genes within the refined interval, the complete sequence of all
exons was analyzed to locate the EMS-induced NC14 mutation. A
single-nucleotide change in SoxN alters glutamine 351, according to
the FlyBase annotated sequence AAF52712.1.
Antibody staining and western blotting
Antibody staining was as described by Dierick and Bejsovec
(Dierick and Bejsovec, 1998).
Mutant chromosomes were maintained over balancer chromosomes marked with
twist-Gal4 UAS-green fluorescent protein (GFP), to identify
homozygotes by their failure to express GFP. Anti-En antibody was used at
1:50, and anti-Arm and anti-GFP at 1:500 [anti-En and anti-Arm from the
Developmental Studies Hybridoma Bank (DSHB), anti-GFP from Chemicon].
Immunoblots were performed as previously described
(Chao et al., 2003) with
embryos that were hand-selected for the appropriate GFP genotype. Filters were
stained with anti-Arm protein (DSHB) at 1:100 and anti-Tubulin at 1:5000 (Lab
Vision). Cross-reacting proteins were detected and quantified using the
Odyssey infrared imaging system and reagents (Li-Cor Technologies).
Cell-culture conditions and luciferase assays
The intronless SoxN sequence was cloned by PCR from genomic fly
DNA to create pcDNA-SoxNflag. pcDNA-Tcf4myc and pcDNA-beta-catphos were
gifts from P. Casey (Duke University Medical Center, Durham, NC). TOP-L
and FOP-L were generated by deleting the 1.681 kb XbaI
fragment containing most of the luciferase gene from the TOPflash- and
FOPflash-reporter plasmids (Upstate). HEK293T cells were grown in DMEM medium
supplemented with 10% FBS. TOPflash-reporter plasmid (0.1 µg), 0.25 ng
phRG-B (Promega) internal control and a total of 0.5 µg expression plasmid
was used for each transfection with lipofectamine2000 (Invitrogen). TOPflash
expression was induced by co-transfection with 0.25 µg of
pcDNA-beta-catphos or by culturing cells in conditioned media from a
Wnt3A stably transfected L-cell line (gift from P. Casey). Un-induced cultures
were similarly treated using conditioned media from untransfected L-cells.
Luciferase activities were determined 24-48 hours post-transfection using the
Dual-luciferase reporter assay system (Promega). Duplicate transfections were
made for each experiment and at least two independent experiments were
performed.
For immunoprecipitation, antibody-conjugated beads were prepared by mixing
20 µl ProtG beads (Zymed) with 1 µl antibody [either anti-Myc (Cell
Signaling) or anti-Flag M2 (Sigma)] in 1 ml PBS with protease inhibitors
(Roche mini tablets) at 4°C overnight. Cell lysates were added to the
prepared beads and treated following standard protocols
(Sambrook et al., 1989).
Immunoblots were stained with anti-Myc (1:10,000) or anti-Flag M2 (1:10,000)
and anti-beta-catenin (1:1000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Hamada et al.,
1999; McCartney et al.,
1999). Only two known fly genes produce an excess-naked-cuticle
phenotype in zygotically mutant embryos: the original segment polarity gene,
naked cuticle (Jürgens et
al., 1984), and the recently characterized RacGap1
ortholog, RacGap50C (Jones and
Bejsovec, 2005). Although the exact mechanisms of action for these
two cytosolic gene products are still not clear, in both cases the phenotype
reflects inappropriate activation of the Wg pathway.
The NC14 zygotic phenotype also indicates ectopic Wg pathway
activity. We recombined the NC14 mutation onto a chromosome bearing a
null allele of wg, wgCX4, and found that it was also
suppressed, although to a lesser degree than the hypomorphic allele
(Fig. 1C,D). Thus,
NC14 rescues segmental patterning in a ligand-independent fashion.
Rescue could also be detected at the molecular level. The epidermal expression
of engrailed (en), in stripes of cells immediately posterior
to the wg-expressing stripes of cells, is dependent on Wg signal
transduction (DiNardo et al.,
1988; Martinez Arias et al.,
1988). In the absence of Wg activity, epidermal en
expression was initiated normally by pair-rule-gene transcription factors but
decays by developmental stage 9. In wgCX4, NC14
double-mutant embryos, however, some epidermal en expression
continued to be detected throughout later stages of development
(Fig. 1E,F). This derepression
of Wg target gene expression is reminiscent of the derepression observed in
wgCX4; gro and wgCX4; Tcf
double-mutant embryos (Cavallo et al.,
1998). On its own, the NC14 mutation produced an
expansion of the en expression domain
(Fig. 2C,D). Wild-type stage-10
embryos expressed en in stripes that ranged from 2 to 3 cells wide,
whereas NC14 homozygotes consistently showed stripes ranging from
between 3 and 5 cells wide. This degree of expansion is similar to that
produced by hyperactivating the Wg pathway within each segment, either by
overexpressing wild-type Wg or by removing a known negative regulator, such as
naked cuticle (Noordermeer et
al., 1992). The NC14 mutation did not affect wg
gene expression (data not shown) and partially restored target gene expression
in a wg-null mutant (Fig.
1F); therefore, it is likely to disrupt a negative regulator
downstream of Wg receptor activation.
To determine the molecular identity of the mutated gene, we subjected the
NC14 mutant chromosome to higher-resolution mapping techniques,
including deficiency analysis and male site-specific recombination. In testing
deficiencies that span the meiotic-map position of NC14, we
identified several deficiencies that fail to complement the embryonic
lethality of the mutation. Embryos transheterozygous for NC14 and
these deficiencies show an epidermal pattern with excess naked cuticle,
identical to that of NC14-homozygous embryos (compare
Fig. 3A with
Fig. 2B). Thus, the
NC14 allele appears to be amorphic according to the classical
definition for null alleles (Muller,
1932).
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) was used to force recombination at the position of
molecularly defined P-element insertions. Candidate genes within the refined
interval were then sequenced and a nonsense change at glutamine 351 in the
SoxN gene was found in the NC14 line. We performed
complementation tests with existing SoxN mutations, which were
isolated in a screen for nervous system disruption
(Seeger et al., 1993;
Buescher et al., 2002), and
found that NC14 was allelic with these mutations. We therefore refer
to the NC14 mutation as SoxNNC14. A cuticle
pattern defect has been noted previously for SoxN mutants
(Buescher et al., 2002), but
it has not been characterized in any detail. Our finding suggests that
SoxN plays a role in regulating Wg pathway activity in addition to
its role in neuroblast formation. Wg is known to control cell-fate choice in
the developing nervous system (Chu-LaGraff
and Doe, 1993), and, therefore, some of the neuronal defects of
the SoxN mutants may be a secondary consequence of ectopic Wg pathway
activation. However, the effects of SoxN loss of function on
neuroblast formation and specification
(Buescher et al., 2002;
Overton et al., 2002) occur at
earlier times and are more severe than those observed for ectopic Wg
expression (Bhat, 1996),
indicating that SoxN is likely to have separate roles in the two
processes.
SoxN influences epidermal patterning
The patterning defect of SoxNNC14 mutant embryos can be
rescued by the expression of a UAS-SoxN transgene
(Fig. 2E), confirming that
SoxN is the gene responsible for the mutant phenotype. Although
SoxN expression shows a pattern of ectodermal stripes during the late
stages of embryogenesis (Cremazy et al.,
2000; Buescher et al.,
2002), rescue of SoxNNC14 pattern defects can
be achieved by uniform expression with either the arm-Gal4 or
E22C-Gal4 driver lines. Therefore, segmentally striped expression of
SoxN is not required for its role in regulating Wg pathway activity.
There did not appear to be a significant maternal contribution of
SoxN (Fig. 3B).
Embryos derived from homozygous mutant germline clones showed cuticle pattern
defects indistinguishable from the zygotic mutant embryos
(Fig. 3B). Likewise, homozygous
mutant clones of adult tissue did not show any evidence of disrupted pattern,
indicating that SoxN does not play a significant role in regulating
Wg signal transduction in the imaginal disc (data not shown). We also tested
for possible redundancy of SoxN with Dichaete (D),
a second closely related SoxB-class gene in the fly genome
(Nambu and Nambu, 1996;
Russell et al., 1996). This
gene has been found to function redundantly with SoxN in patterning
the embryonic nervous system (Overton et
al., 2002), but did not appear to influence the role of
SoxN in Wg signaling (see Fig. S1 in the supplementary material).
Driving high levels of wild-type SoxN can produce profound disruptions in embryonic patterning, without affecting wg expression or Arm stability (see Fig. S2 in the supplementary material). In otherwise wild-type fly embryos, ectopic SoxN interfered with the normal specification of naked cuticle, resulting in denticles within the naked zone (Fig. 2F). The cuticle defects produced by SoxN overexpression correlated with an inappropriate repression of en expression (Fig. 2G,H), again supporting a role for SoxN in the negative regulation of target gene expression. Segmental patterning on the dorsal surface was often more severely disrupted than on the ventral (Fig. 2F,H), leading to curvature of the cuticle. Loss of SoxN function also had variable and mild effects on dorsal cuticle patterning (Fig. 3C).
SoxN overexpression phenotypes are variable and dose sensitive. Driving UAS-SoxN expression with the strong epidermis-specific E22C-Gal4 produced stronger pattern disruptions and greater embryonic lethality than did driving UAS-SoxN with the more widely expressed but less potent arm-Gal4 driver (Table 1). In both cases, those embryos that hatched often survived to become normal adults, suggesting that ectopic SoxN plays no further role beyond embryogenesis. Consistent with this idea, expressing ectopic UAS-SoxN in the imaginal disc with a dpp-Gal4 driver did not create any apparent pattern disruption in the adult, although it did diminish viability somewhat (Table 1). By contrast, dpp-Gal4-driven expression of the dominant-negative form of Tcf, a known repressor of Wg target gene expression, disrupts adult body pattern and results in complete pupal lethality (Table 1).
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). SoxNGA1192 in trans with the
deficiency produces the same extent of excess naked cuticle as does
SoxNNC14 in trans with the deficiency, indicating similar
allele strength (see Fig. S1 in the supplementary material). We routinely show
SoxNNC14 because this mutant chromosome was extensively
recombined to remove extraneous mutations. During random EMS mutagenesis, such
as that in which the NC14 allele was isolated, multiple mutations may
be induced on a chromosome and these will homozygose along with the mutation
of interest. Therefore, accurate phenotypic analysis requires development of a
stock where only the gene of interest is mutationally disrupted. We know that
the SoxNNC14 lesion is the only lethal mutation on the
chromosome, because homozygous viable recombinants to the left and right of
the lesion were recovered during male site-specific recombination mapping.
We tested SoxN mutations with strong
(Fig. 3E,F;
Table 2) and weak alleles of
arm, and found that, in both cases, the arm; SoxN double
mutants showed the `lawn of denticles' phenotype typical of arm
mutants. Double mutants could be distinguished from arm single
mutants among the progeny of this cross because the SoxN mutation
disrupted dorsal pattern and reduced the size of arm mutant embryos.
Thus, SoxN slightly enhances the severity of the effects of
arm on overall body patterning. Because arm gene activity is
required for the specification of excess naked cuticle observed in
SoxN mutants, SoxN must act upstream or in parallel with Arm.
However, Arm protein levels are not artificially stabilized in SoxN
mutant embryos (Fig. 2I-K) like
they are in other zygotic-mutant conditions that produce excess naked cuticle,
such as RacGap50C mutants (Jones
and Bejsovec, 2005). SoxN mutant embryos showed stripes
of Arm accumulation similar to those of wild-type siblings
(Fig. 2I,J), indicating that
the signal transduction machinery upstream of Arm functions properly and that
epidermal cells respond normally to the striped production of Wg signal. This
is true for both the SoxNNC14 and
SoxNGA1192 (shown) alleles. Thus, SoxN is a
unique zygotically acting mutation that hyperactivates the Wg pathway without
affecting Arm stability.
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).
Surprisingly, not only was the SoxN mutant phenotype epistatic to
Tcf loss of function, but loss of Tcf enhanced the naked
cuticle specification in SoxN mutant embryos. SoxN; Tcf
double mutants showed more extensive naked cuticle than did the SoxN
single mutant (Fig. 3H,
Fig. 2B,
Table 2). These data suggest
that the repressive form of Tcf may act synergistically with SoxN to
downregulate Wg pathway activity. Consistent with this idea, we found that
reducing the dose of gro also enhanced the naked cuticle
specification in SoxN mutant embryos
(Fig. 2B,
Fig. 3G). This effect was only
observed when the gro allele was introduced from the mother. Previous
work has shown that gro suppression of wg loss of function
is also strictly a maternal phenotype
(Cavallo et al., 1998).
Conversely, reducing maternal gro reduced the severity of pattern
disruptions caused by ectopic UAS-SoxN expression
(Fig. 2F,
Fig. 3D). Thus, maternally
provided Gro+ functions as a Wg target co-repressor whether or not
the SoxN gene product is present, and appears to increase the
repressor activity of SoxN in a dose-dependent fashion.
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; Cavallo
et al., 1998). Therefore, it strongly and constitutively represses
Wg target gene expression. Lowering the dose of SoxN partially
rescues the segmental pattern disruptions caused by
TcfDN-transgene expression, and also substantially rescues
head cuticle defects and increases the overall size of the body
(Fig. 3J,K). This indicates
that wild-type SoxN activity contributes to the repressor activity of Tcf in a
dose-dependent fashion. The idea that SoxN interacts with Tcf is further supported by overexpression experiments involving the wild-type forms of both molecules. Overexpressing wild-type Tcf enhanced the repressive capacity of overexpressed wild-type SoxN. This was particularly obvious when the UAS transgenes were driven ubiquitously at lower levels with the arm-Gal4 driver. Under these conditions, UAS-Tcf had no effect on either en expression or on cuticle pattern (Fig. 4A,B) and embryos typically hatched and grew to adulthood. arm-Gal4-driven UAS-SoxN showed an only modest narrowing of en expression domains (Fig. 4C) and few ectopic denticles (Fig. 4D). When both transgenes were driven simultaneously, en expression was more dramatically narrowed (Fig. 4E) and the cuticle pattern was more disrupted, both ventrally and dorsally (Fig. 4F). By calculating the rates of embryonic lethality in the transgenic crosses, we found that the synergy between SoxN and Tcf cannot be explained as simple additivity (Table 1). With either the arm-Gal4 or E22C-Gal4 embryonic drivers, fewer embryos co-expressing SoxN and Tcf survived than those where SoxN alone is overexpressed. With the potent E22C-Gal4 epidermal driver, the embryonic lethality rate of double-transgenic embryos approached the rate of embryos expressing dominant-negative Tcf. Co-expression of Tcf with SoxN also affected adult patterning. The expression of either transgene individually in the imaginal disc with dpp-Gal4 had modest effects on adult eclosion rates. Combining the transgenes produced pupal lethality as profound as that of the dominant-negative Tcf (Table 1).
These co-expression results argue against a simple model in which SoxN downregulates target expression by competing with Tcf for Tcf-binding sites. In this simple model, overexpressing wild-type Tcf should reduce the severity of ectopic wild-type SoxN; however, instead we observed an increase in severity. Furthermore, SoxN did not appear to act by sequestering Arm away from Tcf. Co-expressing arm with UAS-SoxN did not affect the SoxN overexpression phenotype (n=467), nor did reducing maternal arm dose (n=553). Likewise, we observed no evidence of arm dosage effects on the SoxN mutant phenotype (Table 2).
SoxN represses mammalian Wnt signal transduction
To determine whether the relationship between SoxN and
Tcf is conserved in vertebrates, we made use of the TOPflash (Tcf
optimal binding sites) reporter system expressed in human embryonic kidney
293T (HEK293T) cells (Korinek et al.,
1998; Ishitani et al.,
1999). We found that SoxN expression reproducibly
diminished Tcfmediated transcription in a dose-dependent fashion
(Fig. 5A), comparable with
other known negative regulators of Wnt gene expression, such as gro
(Cavallo et al., 1998). This
demonstrates that the fly SoxN protein interacts with vertebrate pathway
components to antagonize Wnt-stimulated gene expression in mammalian cells.
Similar repression is observed whether TOPflash is activated with
Wnt-conditioned medium or by co-transfection with a constitutively active
beta-catenin. Thus, artificially elevated beta-catenin levels do not affect
Sox-mediated repression.
We tested whether the addition of extra Tcf-binding sites interferes with
the SoxN repression of TOPflash-reporter activity. The TOPflash plasmid was
altered to delete the luciferase structural gene
(Fig. 5B). We made the same
change in FOPflash (far from optimal), which has mutated Tcf-binding sites
(Korinek et al., 1998;
Ishitani et al., 1999), in
order for it to serve as a negative control. TOP-L competitor DNA,
added at levels in excess of 2.5 times the amount of TOPflash, reduced
reporter activity, whereas FOP-L competitor did not
(Fig. 5C). This indicates that
the intact Tcfbinding sites in the TOP-L competitor can titrate
endogenous Tcf in HEK293T cells. If SoxN preferentially binds to Tcf consensus
sites, we would expect to see a reversal of SoxN-mediated repression with
TOP-L but not with FOP-L. To sensitize the assay, we used 10 ng
of SoxN, which provides an intermediate level of repression, and added
competitor DNA at 2.5 times the amount of TOPflash reporter. Under these
conditions, SoxN is limiting, but endogenous Tcf is not substantially
compromised. We found that co-transfecting with either plasmid did not affect
SoxN repression in the TOPflash assay (Fig.
5D). This suggests that SoxN does not act by directly competing
with Tcf for its consensus binding sequences.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), XSox3,
XSox17 and XSox17ß (Zorn et
al., 1999) - as well as Chibby, a conserved nuclear factor
(Takemaru et al., 2003),
antagonize Wg/Wnt signaling by binding to Arm/beta-catenin and preventing it
from partnering with Tcf to activate target gene expression. SoxN, however,
did not bind beta-catenin in cell-culture assays, and does not share strong
homology with the C-terminal sequences through which vertebrate Sox proteins
bind this protein. Furthermore, we find that SoxN function is not influenced
by Arm levels. No difference was observed in SoxN-mediated TOPflash repression
when cells were induced by co-transfection with a constitutively stabilized
beta-catenin versus with Wnt-induced medium. Instead, both our TOPflash and
our genetic experiments indicate that SoxN function depends on Tcf and Gro,
its co-repressor.
One way to explain our observations is that SoxN contributes to the
assembly or stability of the Tcf repressor complex on DNA
(Fig. 6). The
consensus-sequence recognition for HMG domains in the Sox and Tcf families is
reported to be similar (reviewed in
Clevers and van de Wetering,
1997; Kamachi et al.,
2000; Wilson and Koopman,
2002), although XSox3 and XSox17ß fail to bind a consensus
Tcf DNA sequence (Zorn et al.,
1999; Zhang et al.,
2003). We show that SoxN does not compete for Tcf-binding sites as
a means of repressing target gene transcription, but our data support a model
in which SoxN might bind DNA elsewhere or might bind Tcf sites transiently to
initiate or stabilize the assembly of a repressor complex.
A similar model may explain the results from Xenopus that showed
that XSox3-mediated repression does not require interaction between XSox3 and
beta-catenin (Zhang et al.,
2003). XSox3 strongly interferes with dorsal fate specification in
Xenopus embryos and represses TOPflash-reporter activity in vitro.
HMG-domain mutations render XSox3 inactive in embryos without affecting its
interaction with beta-catenin or its repression in TOPflash assays. Thus, it
is the DNA-binding domain, not the beta-catenin-interacting C-terminus, that
is relevant to its in vivo function in dorsal determination in
Xenopus. XSox3 represses the expression of the dorsal-specific
Nodal-related gene Xnr5 through optimal core binding sequences
adjacent to and partially overlapping with Tcf sites in the Xnr5
promoter (Zhang et al., 2003).
By contrast, the fly SoxN shows no discrepancy between its behavior in
TOPflash assays and its in vivo effects. This suggests that the synthetic
Tcf-binding sites arranged in the TOPflash-reporter plasmid are sufficient to
support SoxN repressor function.
Because adding Tcf-site competitor DNA does not diminish the repressive
capacity of limiting amounts of SoxN (Fig.
5A-D), the role of SoxN in repression does not appear to be
stoichiometric. Therefore, we favor the idea that Sox proteins may act in a
catalytic fashion during repressor-complex assembly at Wnt target gene
promoters, rather than forming a structural part of the repressor complex
itself. We have been unable to detect direct binding of SoxN with either Tcf,
Gro or Arm, raising the possibility that SoxN interacts with some as yet
unidentified protein that chaperones assembly of the repressor complex. A
SoxN-binding cofactor, SNCF, was previously identified in Drosophila
(Bonneaud et al., 2003), but
this gene is expressed only in pre-gastrulation embryos. Because Wg signaling
occurs exclusively post-gastrulation, and specification of naked cuticle
begins more than 4 hours after gastrulation
(Bejsovec and Martinez Arias,
1991), we do not believe that SNCF is a likely candidate for
mediating this aspect of SoxN function. Rather, it is likely to play a role in
the neuronal specification events promoted by SoxN at earlier stages of
embryogenesis.
We find it curious that uniformly overexpressed SoxN represses Wg
signal transduction in dorsal epidermal cells more severely than in ventral
cells. This effect is evident in both cuticle pattern elements and in
en expression, and is reminiscent of defects observed in the
`transport-defective' class of wg mutant alleles, which includes
wgNE2. These mutations restrict Wg-ligand movement
ventrally to promote only local signaling response while simultaneously
abolishing all dorsal signaling (Dierick
and Bejsovec, 1998), suggesting a fundamental difference in
ventral and dorsal cell response. Perhaps it is not a coincidence that the
NC14 mutation was isolated in the wgNE2 mutant
background. Further analysis of SoxN function may help us to
determine the molecular basis for dorsoventral differences in Wg signal
transduction.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/989/DC1
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ACKNOWLEDGMENTS |
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