Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada
* Author for correspondence (e-mail: everheye{at}sfu.ca)
Accepted 10 March 2004
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SUMMARY |
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Key words: NLK, Wg, Wing development
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Introduction |
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In addition to extrinsic regulatory factors, inducible feedback loops have
been found for most conserved signal transduction pathways controlling
development (Freeman, 2000).
In Drosophila, two inducible inhibitors of Wg signaling have been
described that target distinct steps in the pathway. naked cuticle
(nkd) encodes a cytoplasmic protein that binds to Dsh and blocks
accumulation of Arm in response to Wg signaling during embryonic patterning
and eye development (Rousset et al.,
2001
; Zeng et al.,
2000
). Conversely, wingful (wf) encodes a
secreted extracellular feedback inhibitor that acts non-autonomously during
larval imaginal disc development to inhibit Wg
(Gerlitz and Basler,
2002
).
Wg function is required throughout Drosophila development in a
wide range of patterning events (Cadigan
and Nusse, 1997). During wing development, Wg signaling plays at
least two distinct roles. Early reductions of wg result in
wing-to-notum transformations, indicating a requirement for Wg in defining the
wing blade (Morata and Lawrence,
1977
; Ng et al.,
1996
). Later reductions cause wing margin notching due to tissue
loss, indicating the subsequent role of Wg in specifying the margin and
organizing wing development (Couso et al.,
1994
; Diaz-Benjumea and Cohen,
1995
; Rulifson and Blair,
1995
). In late third larval instar wing imaginal discs, Wg is
expressed in a narrow stripe of three to six cells straddling the dorsoventral
(DV) boundary of the future wing blade
(Baker, 1988
;
Couso et al., 1994
;
Williams et al., 1993
).
Directly adjacent to the stripe, Wg regulates the expression of high-threshold
(or short-range) target genes, including achaete (ac) and
neuralized (Phillips and Whittle,
1993
; Couso and Arias,
1994
; Zecca et al.,
1996
). In addition to these targets of Wg signaling,
Distal-less (Dll) is expressed in a Wg-dependent manner in a
wider domain radiating from the thin DV stripe
(Zecca et al., 1996
).
Drosophila nemo (nmo) was first identified as a gene
required for epithelial planar polarity (EPP) during ommatidial development, a
process known to involve the Frizzled (Fz) receptor and which is proposed to
signal through a non-canonical Wnt pathway
(Choi and Benzer, 1994;
Mlodzik, 2002
). Subsequent
analysis has shown that Nemo functions in multiple tissues and has diverse
roles in development. In addition to its effect on eye polarity, we have found
that disruption of nmo results in changes in wing shape and size,
wing vein specification, fertility and viability
(Verheyen et al., 2001
).
nmo is essential for embryonic development as loss of maternal and
zygotic nmo results in embryonic lethality characterized by
patterning defects in the head and ventral denticle belts as well as
disruption of apoptosis (Mirkovic et al.,
2002
).
Nemo is the founding member of an evolutionarily conserved family of
proline-directed serine/threonine protein kinases (referred to as Nemo-like
kinases, NLKs) that includes the murine and human Nemo-like kinases (Nlk),
C. elegans LIT-1, Fugu rubripes NLK and Xenopus
xNLK (Choi and Benzer, 1994;
Brott et al., 1998
;
Harada et al., 2002
;
Hyodo-Miura et al., 2002
;
Kehrer-Sawatzki et al., 2000
;
Meneghini et al., 1999
;
Rocheleau et al., 1999
). NLKs
can exert an inhibitory effect on the gene regulation activity of TCF/LEF
transcription factors (Ishitani et al.,
1999
; Rocheleau et al.,
1999
; Shin et al.,
1999
). Nlk mediates phosphorylation of TCF and inhibits the
DNA-binding ability of the TCF/ß-catenin complex
(Ishitani et al., 1999
). In a
C. elegans non-canonical pathway, activation of the LIT-1 kinase
requires WRM-1, a ß-catenin-like protein, and leads to phosphorylation of
LIT-1 and WRM-1 and subsequent phosphorylation and inhibition of a nematode
TCF, POP-1 (Rocheleau et al.,
1999
).
NLKs have been found to participate in both canonical and non-canonical Wnt
pathways. In C. elegans, LIT-1 has been found to play roles in cell
polarity and cell fate decisions, two processes regulated by distinct Wnt
pathways (Ishitani et al.,
1999; Meneghini et al.,
1999
; Rocheleau et al.,
1999
). Analysis of NLK function in Xenopus oocyte axis
formation assays has shown that injection of murine Nlk and
xNLK mRNAs can block axis formation and can rescue the axis
duplication induced by ß-catenin or Wnt
(Hyodo-Miura et al., 2002
;
Ishitani et al., 1999
).
Consistent with these findings, genetic and phenotypic analyses in
Drosophila support the proposed role for Nemo in both canonical and
non-canonical Wnt signaling pathways. In addition to its role in the
non-canonical Fz pathway regulating epithelial planar polarity (EPP) in the
eye, wing and abdomen (Choi and Benzer,
1994; Strutt et al.,
1997
; Verheyen et al.,
2001
), we have previously reported preliminary evidence that
modulating levels of nmo results in phenotypes consistent with a role
as a Wg-antagonist (Verheyen et al.,
2001
).
In this study, we present our thorough study of the role of Nemo in Drosophila canonical Wg signaling. Through detailed genetic analysis we observe that nmo is an antagonist of Wg during larval wing disc development and that Nemo can negatively influence Wg-dependent gene expression. In addition we present evidence that transcription of nmo is induced by high levels of Wg signaling in the developing wing disc. Finally, we show that cellular levels of Armadillo protein can be controlled by Nemo, such that ectopic Nemo leads to reductions in stabilized Arm. Our results indicate that Nemo is an intracellular inducible feedback antagonist of the Wingless signaling pathway that is involved with refining the Wg activity gradient during wing development.
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Materials and methods |
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Clonal analysis
nmo somatic clones were induced using the FLP/FRT method
(Xu and Rubin, 1993). To
induce nmo loss-of-function clones, embryos from the appropriate
crosses were collected for 24 hours and heat shocked at 38°C for 90
minutes at 48 hours of development. The genotypes examined were: for Wg and
Arm staining in nmo clones, y hs-Flp122; nmo FRT
79D/Ubi-GFP FRT79D; for ß-galactosidase staining of
Dll-lacZ in nmoDB24 clones, y hs-Flp122;
Dll-lacZ/+; nmoDB24 FRT 79D/Ubi-GFP FRT79D; and for ß-gal
staining in dsh clones, dshv26/GFP, FRT 18A;
hsFLP38/+; nmo-lacZ/+.
To induce `flip-out' clones ectopically expressing active Arm,
AyGal4.25-UAS-GFP.S65T; nmo-lacZ/TM6B flies were crossed to
UAS-fluarm; hs-Flp flies
(Ito et al., 1997
). To induce
clones of ectopic Nemo expression, AyGal4.25-UAS-GFP.S65T,
UAS-nmoC5-1e flies were crossed to hs-Flp flies. To
induce clones of ectopic Daxin, AyGal4.25-UAS-GFP.S65T; nmo-lacZ/TM6B
flies were crossed to hs-Flp; UAS-Daxin flies.
Immunostaining and in situ hybridization
Dissection of imaginal discs, X-Gal staining and antibody staining were
carried out using the following standard protocols. The antibodies used were:
mouse anti-Wg (1:100) and anti-Armadillo (1:200) concentrated supernatants
from the Developmental Studies Hybridoma Bank; mouse anti-ß-galactosidase
(1:500) from Promega; rabbit anti ß-galactosidase (1:2000) from Cappel.
Secondary antibodies used were: donkey anti-mouse FITC (Jackson Immunolabs),
donkey anti-mouse AlexaFluor 594 (Molecular Probes), donkey anti-rabbit CY3
and FITC (Jackson Immunolabs). All secondary antibodies were used at 1:200
dilutions. In situ hybridization was performed according to Tautz and Pfeiffle
(Tautz and Pfeiffle,
1989).
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Results |
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The nmo-lacZ pattern is reminiscent of the Wg expression pattern
in imaginal discs (Rulifson et al.,
1996). To examine the relationship between the two expression
patterns, we performed double staining for ß-galactosidase and Wg
protein. This staining reveals that nmo expression at the DV boundary
flanks the Wg protein domain in late third instar wing discs
(Fig. 1E-G). Wg protein is
detected in a narrow stripe along the presumptive wing margin
(Fig. 1G) and nmo is
seen in the cells directly adjacent to the Wg-expressing cells
(Fig. 1E). In addition,
nmo is detected in the ring domain overlapping with the Wg inner ring
expression domain that encircles the wing pouch
(Fig. 1F). Such a localization
for nmo is also consistent with the observed defect in adult flies in
which the wing is held away from the body at an angle and may reflect a hinge
defect (Verheyen et al.,
2001
).
nmo antagonizes Wg signaling during wing development
Based on the expression pattern of nmo and data suggesting a role
in Wnt signal transduction, we investigated the role of Nemo in Wg signaling
using a combination of approaches, involving ectopic expression, mutant
analysis, somatic loss-of-function clones and ectopic flip-out misexpression
clones (Brand and Perrimon,
1993; Ito et al.,
1997
; Xu and Rubin,
1993
). Wg is expressed along the presumptive wing margin where it
is required for proneural achaete-scute (AS-C) complex gene
expression and for the formation of margin bristles. Loss of Wg signaling
along the wing margin leads to loss of these margin bristles and the
appearance of notches along the wing margin
(Couso et al., 1994
;
Phillips and Whittle, 1993
;
Rulifson et al., 1996
).
Ectopic expression of UAS-nmo in the wing using either
scalloped-Gal4 (referred to as sd>nmo) or
omb-Gal4 also produces such a wing notching effect
(Fig. 2B, and data not shown),
suggesting Nemo plays an antagonistic role in the pathway. A similar wing
notching phenotype is seen when either 71B-Gal4 or 69B-Gal4
is used to drive expression of the Wg inhibitor Daxin
(Hamada et al., 1999
;
Willert et al., 1999
). The
observed wing margin loss seen in sd>nmo flies is completely
suppressed when flies are heterozygous for the zw3m11
loss-of-function allele (Fig.
2C), consistent with the antagonistic role that Zw3 plays in Wg
signaling and with the speculation that the effect of nmo is due to
blocking the action of Wg.
To extend this study, we examined whether loss of nmo or
ectopically expressed Nemo is able to suppress defects caused by
overexpression of Wg pathway components. Ectopic expression of Dfz2N, a
dominant-negative form of the Drosophila Frizzled 2 receptor
(Zhang and Carthew, 1998)
using the sd-Gal4 driver induces a tiny wing phenotype characterized
by loss of the wing margin and significant amounts of wing blade
(Fig. 2E). Flies homozygous for
nmoDB24, a putative null allele of nmo, have a
broader, shorter wing than wild type and ectopic vein material near
longitudinal vein 2 and 5 and emanating from the posterior cross vein
(Fig. 2D) (D. Bessette and
E.M.V., unpublished). The sd>Dfz2N phenotype is significantly
suppressed when flies are homozygous for nmoDB24,
resulting in restoration of most wing margin structures as well as wing blade
tissue (Fig. 2F). Furthermore,
we found that nmoDB24 also suppresses the effects of Daxin
in a dose-sensitive manner (Fig.
2G-L). sd>Daxin causes wing-to-notum transformations
(Fig. 2G) that can be rescued
to a small wing by heterozygosity for nmoDB24
(Fig. 2H). Stronger suppression
is detected in homozygous nmoDB24 flies, in which the
ectopically produced nota are completely suppressed and the wing blade is
partially restored, particularly in the anterior wing margin
(Fig. 2I). The same
dose-sensitive suppression is observed when the dorsally expressed
ap-Gal4 driver was used to drive Daxin. ap>Daxin induces
a tiny blistered wing pouch (Fig.
2J). Heterozygosity for nmoDB24 in this
background partially rescues the pouch defect
(Fig. 2K), while
nmoDB24 homozygosity strongly rescues the wing blisters
and abnormal appearance (Fig.
2L). These data suggest that the block in Wg signaling caused by
ectopic Daxin can be suppressed by the absence of nmo function.
Additional evidence supporting the involvement of Nemo as a negative player
in the Wg pathway comes from examining interactions with Arm.
UAS-fluarm encodes an N-terminally truncated,
constitutively active form of Arm
(Tolwinski and Wieschaus,
2001
; Zecca et al.,
1996
). Using 71B-Gal4 to drive
UAS-flu
arm causes a very abnormal wing
(Fig. 2M) characterized by
excess margin bristles throughout the wing blade, loss of veins and a smaller
crumpled wing blade, similar to the abnormal wing seen with ectopic expression
of LEF-1 (Riese et al., 1997
).
While 71B>nmo induces no visible wing defects
(Fig. 2N), ectopic expression
of nmo is able to suppress the 71B>flu
arm
wing phenotype by restoring the size of the wing blade, reducing ectopic
bristles and wing blistering (Fig.
2O).
In addition to interactions in wing patterning, nmo antagonizes Wg
signaling in the sensory bristles of the notum. ap>nmo flies
display a loss of notum bristles (Fig.
3B). This phenotype is opposite to that seen upon ectopic
activation of Wg signaling (Phillips et
al., 1999; Riese et al.,
1997
; Simpson and Carteret,
1989
). The ap>nmo bristle loss phenotype is suppressed
by heterozygosity for zw3m11
(Fig. 3C) and enhanced by
co-expression of Daxin, resulting in loss of all scutellar bristles
(Fig. 3D). ap>nmo
flies also display an abnormal wing phenotype in which the wing blades do not
appose properly, forming a large blister
(Fig. 3E). Heterozygosity for
zw3m11 suppresses this effect, resulting in a significant
rescue of wing morphology (Fig.
3F).
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nmo autonomously suppresses Wg-dependent gene expression
In the wing disc, Wg signaling positively regulates Distal-less
(Dll) expression (Zecca et al.,
1996). Dll is expressed in a domain overlying but wider than the
Wg DV expression domain and can be induced by ectopic Wg signaling
(Fig. 4A)
(Zecca et al., 1996
). Thus the
normal pattern of Dll is governed by Wg signaling and Dll expression can be
used to monitor the activity of the Wg pathway. As our genetic analysis
strongly indicates that Nemo antagonizes Wg signaling, we examined whether
modulation of Nemo could affect Dll expression. We found that ectopic
expression of nmo was able to suppress the expression of
Dll-lacZ in an ap>nmo background
(Fig. 4B).
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wg gene expression is not regulated by nmo
As Nemo inhibits Wg-dependent gene expression, we were interested in
whether Nemo played any negative role in regulating wg expression
itself. In embryos, wg gene expression is positively regulated in an
autocrine fashion in response to Wg signaling
(Hooper, 1994), whereas in
wing discs Wg acts to repress wg expression in neighboring cells
(Rulifson and Blair, 1995
). Wg
expression in nmo mutant somatic clones was examined and no change of
Wg protein staining was detected (Fig.
5A-C). We also generated somatic flip-out clones ectopically
expressing nmo in wing discs. Similar to what was found in mutant
clones, no alterations in Wg expression were observed in flip-out clones in
wing discs ectopically expressing Nemo
(Fig. 5D-F).
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nmo is a novel Wg target gene
Considering that the expression pattern of nmo flanks that of Wg
in wing imaginal discs, we speculated that the expression of nmo may
be regulated by Wg signaling. We first examined the effect of ectopic Wg
pathway activation on nmo-lacZ staining. Expression of activated
UAS-fluarm using vg-Gal4 causes high levels
of Wg pathway activation and leads to ectopic nmo-lacZ expression
along the vg-Gal4 expression domain
(Fig. 6A,B)
(Zecca et al., 1996
). The two
DV boundary stripes become less defined and appear to expand
(Fig. 6B, compare to
Fig. 1C). Similarly,
dpp>flu
arm induces nmo-lacZ expression
along the AP boundary (Fig.
6C). These results indicate that activation of the Wg pathway can
lead to nmo gene expression.
|
To determine whether loss of Wg signaling activity could also affect
nmo expression, we generated UAS-Axin flip-out clones and
examined the effects on nmo-lacZ staining. In such clones, marked by
GFP staining (Fig. 6G,H),
nmo expression is suppressed (Fig.
6H,I) in both regions of high (arrow in
Fig. 6I) and low (arrowhead in
Fig. 6I) expression. We then
examined somatic clones homozygous mutant for dishevelled
(Fig. 6J,K) and we find a
cell-autonomous inhibition of nmo expression
(Fig. 6K,L). In all cases, we
observe inhibition of not only the high levels of DV boundary nmo but
also the low level ubiquitous staining within the wing pouch. We also examined
the effect of ectopic expression of UAS-Fz2N and found that
vg>Fz2N wing discs display a loss of nmo
staining at the DV boundary which is similar to the inhibitory effect of
UAS-Fz2N on other Wg downstream genes such as the DV boundary marker
vg-lacZ (data not shown) (Zhang
and Carthew, 1998). All of these results taken together confirm
that activation of endogenous Wg signaling results in nmo expression,
and that nmo is a bona fide Wg target gene.
Nemo can affect Arm stabilization
The localization of nmo in third instar wing discs is very
reminiscent of the pattern of stabilized Arm protein observed after Wg pathway
activation (Peifer et al.,
1991; Mohit et al.,
2003
). To examine this more closely, we carried out double
staining to detect nmo gene expression and Arm protein stabilization.
First, we observed that nmo and stabilized Arm co-localize in the
central region of the wing margin (Fig.
7A-C). In addition, we noted that in the anterior region of the
wing margin where nmo expression is elevated, Arm protein levels are
lower, relative to the rest of the margin. Third, we find that Nemo staining
is reduced in the region where the DV and AP boundaries intersect and that
this region shows more stabilized Arm protein.
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Discussion |
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The canonical Wnt pathway also makes use of negative feedback mechanisms.
In murine Wnt signaling, the feedback loops primarily target the activity of
ß-catenin. For example, Tcf1 is a target gene for ß-catenin/Tcf4 in
epithelial cells and is proposed to act as a repressor that counteracts
ß-catenin/Tcf4-mediated gene expression
(Roose et al., 1999).
Spiegelman et al. (Spiegelman et al.,
2000
) have provided evidence that the ß-TrCP protein, the
expression of which is induced by ß-catenin/TCF signaling, targets
ß-catenin for ubiquitination and subsequent degradation
(Spiegelman et al., 2000
). It
has also been shown that expression of Axin2, one of the scaffold proteins in
the inhibitory APC/GSK3ß complex, is also induced by Wnt signaling
(Jho et al., 2002
).
In Drosophila, several examples of Wg feedback inhibition have
been identified. First, it has been shown that Wg downregulates its own
transcription in the wing pouch to narrow the RNA expression domain at the DV
boundary (Rulifson et al.,
1996). Second, Wg signaling can repress the expression of its
receptor Dfz2 in the wg-expressing cells of the wing disc. Wg
regulation of Dfz2 creates a negative feedback loop in which newly secreted Wg
is stabilized only once it moves away from the DV boundary to cells expressing
higher levels of Drosophila Fz2
(Cadigan et al., 1998
). Third,
the Wg target gene naked cuticle (nkd) acts through Dsh to
limit Wg activity (Rousset et al.,
2001
; Zeng et al.,
2000
). Fourth, Wingful (Wf), an extracellular inhibitor of Wg, is
itself induced by Wg signaling (Gerlitz
and Basler, 2002
).
Nemo is an inducible inhibitor of Wg
Our research adds Nemo to this list of inducible antagonists participating
in Wg signaling (Fig. 8). We
show that Nemo antagonizes the Wg signal in wing development, as evidenced by
phenotypic rescue, suppression of Wg-dependent gene expression in discs
ectopically expressing nmo, and ectopic expression of a Wg-dependent
gene in nmo mutant clones.
|
The effect of Nemo on the Wg-dependent reporter gene Dll is
confined to regions of endogenous gene expression. In the absence of
nmo expression, ectopic Dll expression is only seen at
elevated levels within the endogenous expression domain, thus being dependent
on Wg activity. This is in contrast to inhibition of the Dpp pathway by
Brinker (Campbell and Tomlinson,
1999; Jazwinska et al.,
1999
; Minami et al.,
1999
). Brinker acts independently of Dpp in its repression of Dpp
target genes, such that in the absence of both brk and Dpp the target
genes are expressed ectopically (Campbell
and Tomlinson, 1999
). We speculate that the role of Nemo in the Wg
pathway is analogous to the role of Daughters against Dpp (Dad) in Dpp
signaling (Tsuneizumi et al.,
1997
). Dpp induces the expression of dad, which in turn
antagonizes the pathway through an as yet undefined mechanism. These might
include either interactions with the intracellular transducer Mothers against
Dpp (Mad) or with TGFß receptors.
Nemo does not participate in the self-refinement of Wg expression
It is intriguing that Nemo does not play a role in regulating wg
expression; however, this is most probably because of the point of action of
Nemo within the Wg pathway. The self-refinement of wg expression in
the wing is dependent on Dsh but independent of Arm
(Rulifson et al., 1996).
Recent work has raised some questions about the factors involved in Wg
self-refinement, specifically postulating a role for dTCF in this process
(Schweizer et al., 2003
). dTCF
(pan) somatic clones were shown to have elevated Wg protein,
suggesting that TCF plays an active role in repressing Wg gene expression. The
authors, however, indicate that they fail to distinguish between increased
wg gene expression and stabilized Wg protein. Another recent paper
examined regulation of Wg signaling by Twins (tws), a protein
phosphatase subunit, and found that it is required for Arm stabilization
(Bajpai et al., 2004
).
Modulation of tws resulted in aberrant Wg signaling, as monitored by
Dll expression, that are not accompanied by alterations in wg gene
expression. Our data are consistent with the findings of Bajpai et al. and
suggest that the mechanism of wg refinement most probably does not
involve Arm or dTCF. Our genetic analyses support the placement of Nemo at or
below the level of Arm within the pathway. The apparent absence of a role for
Nemo in regulating wg expression contrasts with the other inducible
feedback inhibitors. Modulation of either the extracellular inhibitor Wf or
the Dsh-antagonist Nkd can influence wg gene expression in wing discs
and embryos, respectively (Gerlitz and
Basler, 2002
; Zeng et al.,
2000
). As stated above, neither loss of nor ectopic expression of
nmo during imaginal disc development has an effect on the pattern of
Wg expression.
nmo expression is induced by high levels of Wg signaling
The developing wing is bisected by a narrow stripe of Wg-expressing cells.
Wg protein has a short half-life near the DV boundary, which causes a rapid
decrease in Wg concentration and forms a steep symmetric gradient of the Wg
protein (Cadigan et al.,
1998). Radiating out from the source of Wg, there are three
concentric domains of Wg-dependent gene expression (reviewed by
Martinez Arias, 2003
). First,
a very narrow domain of cells adjacent to the highest concentration of Wg
expresses achaete (ac). Second, Dll is expressed in a median
range domain of Wg and third, a long-range domain expresses vg. Our
results suggest that nmo is a short-range target, like ac,
the activation of which is limited by the high threshold of Wg signal. This
may be the explanation for the very narrow pattern of enriched nmo
expression at the DV boundary and the ring domain and the cell-autonomous
induction of nmo in the ectopic
Arm clones.
If higher levels of Wg protein induce nmo expression, it raises
the question of why nmo is not expressed in DV boundary cells. One
possibility is that there are genes that are expressed between the two stripes
of nmo that prevent its expression. In
Fig. 6B, vg-Gal4,
which is mainly expressed at the DV boundary, drives
UAS-fluarm to induce ectopic nmo expression.
In this case, the ectopic expression of nmo fills the gap between the
two endogenous bands. This observation supports a model in which there is a
suppressor(s) located along the DV boundary to silence nmo
expression. The balance between the Wg signal and the suppressor(s) would
refine nmo expression into two thin stripes flanking the DV boundary.
In the case of ectopic UAS-flu
arm, the Wg signal may
overpower the suppressor, thereby allowing nmo to be expressed at the
boundary. In a similar mechanism, it has been shown that Wg can direct the
expression of ac at the margin but that this expression is prevented,
at least partially, by the activity of Cut
(Couso et al., 1994
).
Although the wing margin, ring expression and low level ubiquitous staining
of nmo in imaginal wing discs reflects regulation by Wg signaling,
the other developmental expression patterns, such as staining in primordia of
wing veins, may reflect regulation by other signaling pathways. For example,
the staining in the wing vein primordia that emerges in late third instar and
the gene expression pattern observed in pupal wings reflects the later role of
nmo in wing vein patterning
(Verheyen et al., 2001), which
may involve interactions with EGFR and TGFß signaling.
In further support that Wg signaling regulates the transcription of
nmo, we find several dTCF consensus binding sites in the 5'
region of the nmo gene which may represent enhancer elements (B.
Andrews and E.M.V., unpublished). Indeed, two sites match 9 out of 11 bp
(GCCTTTGAT) of the T1 site (GCCTTTGATCT) in the dpp BE enhancer that
has been shown both in vitro and in vivo to bind and respond to dTCF
(Yang et al., 2000). The
presence of these sites suggests that the observed transcriptional regulation
of nmo by Wg may involve direct binding to the nmo DNA
sequence by dTCF.
Nemo may target Arm for degradation
As a result of comparing the endogenous expression pattern of nmo
with stabilized Arm, we noticed that the highest levels of Nemo excluded Arm
stabilization, while high levels of Arm were present in cells in which
nmo levels were lower. As Arm protein stabilization is a direct
consequence of Wg pathway activation, we sought to examine whether Nemo may
function to inhibit Wg by promoting Arm destabilization and subsequent
breakdown. Indeed, ectopic expression of Nemo can lead to cell-autonomous
reduction in Arm protein levels. This preliminary result suggests a mechanism
in which Nemo may contribute to the destabilization of Arm that involves the
Axin/APC/GSK3 complex. One explanation to account for such a finding would
concern the interaction with TCF in the nucleus and the role of dTCF as an
anchor for Arm (Behrens et al.,
1996; Tolwinski and Wieschaus,
2001
). Given what is known about NLKs, it is likely that Nemo may
act on the ability of the dTCF/Arm complex to bind DNA and activate
transcription (Ishitani et al.,
1999
). Tolwinksi and Wieschaus (Tolwinksi and Wieschaus, 2001)
propose that dTCF acts as an anchor for Arm in the nucleus. It remains to be
determined how efficient this anchor is and whether there are conditions in
which the interaction may become compromised, such as we see with elevated
Nemo. NLKs have been shown to affect the DNA-binding ability of
TCF/ß-catenin (Ishitani et al.,
1999
). Perhaps in the absence of DNA binding, this complex is less
stable and Arm could be free to shuttle to the cytoplasm where it could
associate with Axin or APC and become degraded
(Henderson and Fagotto, 2002
).
We propose that the ectopic nmo in our assay is leading to
destabilization of the dTCF/Arm/DNA complex and thus causing Arm to exit the
nucleus and be degraded through interaction with Axin, APC and GSK3. The
observation that ectopic expression of full-length Arm cannot induce any
activated Wg phenotypes (Orsulic and
Peifer, 1996
) have been explained by the hypothesis that even
these high levels of protein are not sufficient to overcome the degradation
machinery (Tolwinski and Wieschaus,
2001
). Thus, our finding that there is no elevated Arm in
nmo clones is consistent with an inability to overcome the endogenous
degradation machinery; even though less Nemo could lead to more stabilized DNA
interactions, this would not lead to higher levels of stabilized Arm than is
normally found.
Model for Nemo and NLK function in Wnt/Wg signaling
Studies of homologs of Nemo in other species have provided clues to its
function, although it is still not clear if the same mechanism in used in
Drosophila. Our studies in this paper establish that Drosophila Nemo
does in fact play a negative regulatory role in canonical Wg signaling.
Although nmo was originally identified as playing a role in the
non-canonical Fz pathway that regulates tissue planar polarity, its precise
role in that pathway has not been further defined
(Brown and Freeman, 2003;
Choi and Benzer, 1994
;
Strutt et al., 1997
).
In addition to the findings that NLKs can bind to and phosphorylate TCF and
LEF-1 proteins (Ishitani et al.,
2003) and thereby decrease the DNA-binding affinity of the
TCF/ß-catenin complex, a model is emerging that NLKs regulate multiple
HMG-box containing proteins. Recently, it was shown that Xenopus NLK
(xNLK) binds to a novel HMG-domain containing protein HMG2L1, which can
inhibit Wnt signaling in several assays
(Yamada et al., 2003
). In
addition, xNLK binds to xSox11, another HMG-box containing transcription
factor, and they cooperatively induce neural development in Xenopus
(Hyodo-Miura et al.,
2002
).
Although our results do not directly address the molecular mechanism, we speculate that activated Nemo can inhibit the interaction of the Arm-dTCF complex with DNA. The genetic data presented in this paper support the molecular mechanism that Nemo acts downstream of or at the same level as Arm. Indeed, the finding that increased levels of nmo can block accumulation of Arm is intriguing as it suggests that Nemo may regulate Wg at the level of Arm stabilization and dTCF function. At this point, further biochemical experiments are in progress to address these issues. They should shed light on the exact mechanism of function that allows Nemo to be an inducible antagonist of canonical Wg signaling in Drosophila.
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ACKNOWLEDGMENTS |
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