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First published online 30 January 2008
doi: 10.1242/dev.013805
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Department of Genetics and the Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, NH 03755, USA.
Author for correspondence (e-mail:
yfa{at}dartmouth.edu)
Accepted 11 December 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Adenomatous polyposis coli, Armadillo, Wingless, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Zecca et al., 1996). Wg
elicits its biological effects by binding to the Frizzled and Arrow
co-receptors (Bhanot et al.,
1996; Bhanot et al.,
1999; Chen and Struhl,
1999; Kennerdell and Carthew,
1998; Muller et al.,
1999; Tamai et al.,
2000; Wehrli et al.,
2000). Activation of these receptors results in the stabilization
and nuclear accumulation of Armadillo (Arm), a key transcriptional activator
in the pathway (Riggleman et al.,
1990). In the absence of Wg, Arm is targeted for phosphorylation
and subsequent proteolysis by a destruction complex composed of the
scaffolding protein Axin, the two Adenomatous polyposis coli proteins (Apc1
and Apc2), and two kinases, the glycogen synthase kinase 3 homologue
Zeste-white 3 (Zw3), and casein kinase 1
(Ahmed et al., 1998;
Hamada et al., 1999;
Hayashi et al., 1997;
Liu et al., 2002;
McCartney et al., 1999;
Siegfried et al., 1992;
Willert et al., 1999;
Yanagawa et al., 2002;
Yu et al., 1999). Upon Wg
stimulation, this destruction complex is inactivated, thereby resulting in
increased Arm levels and transcriptional activity
(Peifer et al., 1994;
Siegfried et al., 1994;
Tolwinski et al., 2003).
Previous work indicated that a spatial gradient of Wg activity is crucial
for proper patterning of the wing and leg primordia
(Lecuit and Cohen, 1997;
Neumann and Cohen, 1996;
Struhl and Basler, 1993;
Zecca et al., 1996). A more
recent study reveals that proper eye development also requires precise,
concentration-dependent cellular responses to a gradient of Wg activity
(Tomlinson, 2003). The retina
is composed of approximately 800 ommatidia, each of which contains six outer
(R1-R6) and two inner (R7 and R8) photoreceptors (PRs) (reviewed by
Cook and Desplan, 2001;
Mollereau and Domingos, 2005;
Wolff and Ready, 1993).
Ommatidia can be grouped into three functional categories, pale, yellow, and
dorsal rim area (DRA), which are distinguished by opsin expression and light
sensitivity. The outer PRs in each category express the same opsin,
Rhodopsin 1 (Rh1), whereas the inner PRs express one of four
distinct opsins (Fortini and Rubin,
1990). Inner PRs in pale and yellow ommatidia express Rh3
or Rh4 in R7, and Rh5 or Rh6 in R8, respectively,
allowing for color discrimination. By contrast, all inner PRs within DRA
ommatidia are characterized by expression of Rh3 and the
transcription factor homothorax (hth), and by an increased
diameter of their light-sensing organelles, or rhabdomeres
(Fortini and Rubin, 1990;
Tomlinson, 2003;
Wernet et al., 2003). The DRA
ommatidia function as polarized light sensors, and are spatially restricted to
the two outermost rows at the dorsal margin of the retina, extending up to the
dorsoventral equator.
Wg is required for proper patterning of the peripheral retina, and is
expressed within a ring of cells in the presumptive head capsule that
surrounds the retina. The spread of Wg from the head capsule to the retina
results in a gradient of Wg morphogen activity that specifies three distinct
fates in the peripheral ommatidia
(Tomlinson, 2003)
(Fig. 1A). The highest levels
of Wg, found at the very perimeter of the eye, induce apoptosis of all
photoreceptors at the retinal edge, leaving behind a peripheral rim of pigment
cells (Lin et al., 2004;
Tomlinson, 2003). Intermediate
Wg levels, found just inside the pigment rim, specify the DRA ommatidia
(Tomlinson, 2003;
Wernet et al., 2003). Even
lower Wg levels are sufficient to induce the formation of ommatidia that lack
bristles, which are restricted to the three outermost rows of the retina
(Cadigan et al., 2002;
Tomlinson, 2003).
How is a gradient of Wg activity translated into quantitatively distinct
levels of Arm signaling that induce qualitatively distinct cellular responses?
Specifically, how do the different components in the destruction complex
contribute to the level of Arm signaling? Biochemical studies have indicated
that Axin levels are approximately 5000-fold lower than the level of other
members of the destruction complex, and have led to the model that Axin is the
only limiting component, whereas Apc is present in vast excess
(Lee et al., 2003;
Salic et al., 2000). To
address this model, we examined how the reduction of Apc activity to different
degrees affects Arm signaling, both in the absence of Wg, and within the Wg
gradient. We assayed three concentration-dependent readouts of Arm signaling
in PRs: DRA fate specification, shortening of PR length, and apoptosis, which
are induced by progressively higher levels of Arm signaling. We find that both
Apc1 and Apc2 negatively regulate Arm signaling in photoreceptors, but that
the relative contribution of Apc1 is much greater than that of Apc2.
Unexpectedly, we also find that a less than twofold reduction in total Apc
activity, achieved by loss of Apc2, decreases the effective threshold at which
Wg elicits a cellular response, thereby resulting in ectopic responses that
are spatially restricted to regions with low Wg concentration. These results
indicate that within the range of the Wg gradient, Apc activity is not present
in vast excess, but instead is near the minimal level required for accurate
patterning.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In homozygous Apc279 mutants,
loss of mRpS24 activity induces lethality. Therefore, to obtain
viable Apc279 mutant adults, a P[mRpS24]
transgene (Takacs et al.,
2008) was recombined onto the Apc279 mutant
chromosome. Apc233 has a deletion of 1738 nucleotides that
includes sequences encoding the first 349 amino acids, extending to the fifth
Armadillo repeat (Takacs et al.,
2008).
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),
P[Apc213.5] (Ahmed et
al., 2002), svp07482-PZ [Heberlein et al.
(Heberlein et al., 1991);
Bloomington Drosophila Stock Center (BDSC)], Rh3-lacZ
(Fortini and Rubin, 1990)
(BDSC), UAS-arm.Exel (encoding full-length Arm, Exelixis, BDSC),
UAS-armS10 (Pai et
al., 1997), elav-GAL4
(Lin and Goodman, 1994)
(BDSC), GMR-Gal4 12, line MF815
(Freeman, 1996), long
GMR-Gal4 and short GMR-Gal4
(Wernet et al., 2003),
FRT82B arm-lacZ [Vincent et al.
(Vincent et al., 1994);
provided by J. Treisman, Skirball Institute, New York], eyeless-FLP
[Newsome et al. (Newsome et al.,
2000); provided by J. Treisman], Df(3L)H99 FRT80B [White
et al. (White et al., 1994);
provided by F. Davidson, National Cancer Institute, Bethesda, MD],
Df(3R)w6 (provided by M. Bienz, MRC Laboratory of Molecular Biology,
Cambridge, UK), UAS-ara
(Gomez-Skarmeta et al., 1996),
piM75C FRT80B (Xu and Rubin,
1993), pygo10
(Parker et al., 2002),
pan13 and panER1
(Brunner et al., 1997), and
P[mRpS24]. Canton S flies were used as wild-type controls.
Generation of mitotic eye clones
Clones of mutant retinal cells were generated by FLP-mediated recombination
(Xu and Rubin, 1993), using
eyeless-FLP (Newsome et al.,
2000). Clones were detected by loss of expression either of an
arm-lacZ transgene in pupal retinas, or of a P[w+] transgene
in adult eyes.
Genotypes for generating mutant eye clones were as follows:
pygo10 mutant clones: eyeless-FLP/+; FRT82B pygo10/FRT82B arm-lacZ;
Apc233 mutant clones: eyeless-FLP/+; FRT82B Apc233/FRT82B arm-lacZ;
Df(3L)H99 mutant clones in the homozygous Apc1Q8 mutant: eyeless-FLP/+; Df(3L)H99 FRT80B Apc1Q8/piM75C FRT80B Apc1Q8.
Immunohistochemistry
Primary antibodies used for immunostaining were guinea pig anti-Apc2 (GP10)
1:12,000 Takacs et al., 2008),
rabbit anti-Apc1 1:400 (Hayashi et al.,
1997), rabbit anti-β-Gal 1:5000 (Cappel), mouse
anti-β-Gal 1:500 (Promega), guinea pig anti-Hth 1:500
(Abu-Shaar et al., 1999),
rabbit anti-Hth 1:1000 (Kurant et al.,
1998), mouse (9F8A9) or rat (7E8A10) anti-Elav 1:10 (Developmental
Studies Hybridoma Bank, DSHB), rabbit anti-cleaved caspase-3 1:100 (Cell
Signaling Technology), mouse anti-Arm 1:10 (N2 7A1, DSHB). Secondary
antibodies were goat or donkey Alexa Fluor 488 or 568 conjugates 1:200
(Molecular Probes), and goat or donkey Cy3 or Cy5 conjugates 1:200 (Jackson
Immunochemicals). Fluorescent images were obtained on a Leica TCS SP UV
confocal microscope.
Pupal retinas were dissected in PBS, then fixed in 4% paraformaldehyde, 10
mM NaH2PO4 (pH 7.2) for 20 minutes, washed with PBS,
0.1% Triton X-100, and incubated in PBS, 0.1% Triton X-100, 10% BSA for 30
minutes at room temperature. For analysis with Apc1 and Arm antibodies, pupal
retinas were fixed using heat/methanol
(Ahmed et al., 1998).
Incubation with primary antibodies was performed at 4°C overnight in BNT
(PBS, 250 mM NaCl, 1% BSA, 1% Tween 20) or, if anti-cleaved caspase 3 was
used, TBS [50 mM Tris-HCl (pH 7.4), 150 mM NaCl]. Incubations with secondary
antibodies were for 2 hours at room temperature, or 30 minutes at room
temperature if retinas were stained with the anti-cleaved caspase-3
antibody.
Histology
Adult eyes were dissected and fixed for whole mount X-Gal staining
(Tomlinson, 2003), or were
embedded in plastic resin (Durcupan, Fluka), sectioned to 1 µm and stained
with Toluidine Blue (Cagan and Ready,
1989; Wolff,
2000).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). PR
apoptosis, as visualized by the presence of an activated caspase, begins at
the posterior edge of the Apc1Q8 null mutant retina by 35
hours after puparium formation (APF), and expands to encompass the entire
retina by 39 hours APF (Fig.
1D-I). An identical onset and duration of caspase expression is
observed in this ectopic apoptosis of all PRs resulting from Apc1 loss and in
the developmentally regulated PR apoptosis induced by high-level Arm signaling
restricted to the retinal periphery (Lin
et al., 2004; Tomlinson,
2003) (Fig. 1B,C;
see also Fig. S1 in the supplementary material). Indeed, both of these retinal
apoptotic responses can be observed simultaneously
(Fig. 1D,F). These data suggest
that ectopic apoptosis of all PRs upon Apc1 loss is induced by high-level Arm
signaling.
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;
Tomlinson, 2003). If either Wg
or Arm is ectopically expressed at intermediate levels, many PRs
inappropriately express hth and adopt a DRA fate
(Tomlinson, 2003;
Wernet et al., 2003).
Conversely, inactivation of any one of several transducers of Wg signaling,
including the cytoplasmic effector Disheveled (Dsh), the co-receptors Arrow
(Arr) and Frizzled (Fz and Fz2; previously known as DFz2), or the
transcriptional co-activator Pygopus (Pygo)
(Kramps et al., 2002;
Parker et al., 2002;
Thompson et al., 2002),
partially disrupts hth expression and DRA fate specification
(Wernet et al., 2003;
Tomlinson, 2003) (see Fig. S2
in the supplementary material). We examined hth expression in the Apc1Q8 null mutant at 20 and 30 hours APF, prior to the time at which high-level Arm signaling induces PR apoptosis. We found a marked expansion in the range of hth expression throughout the dorsal half of the Apc1 mutant retina (Fig. 2A-F). The expanded hth expression is apparent in the posterior retina by 20 hours APF (Fig. 2C,D). By 30 hours APF, ectopic hth expression is present in nearly all inner PRs in the dorsal half of the retina, and extends either up to or one row above the dorsoventral equator (Fig. 2E,F).
This ectopic hth expression suggests that Apc1 mutant PRs
would have adopted a DRA fate had they survived. To test this hypothesis, we
prevented apoptosis in the Apc1Q8 mutant by using a
deficiency that eliminates three cell-death effector genes reaper, head
involution defective (hid) and grim
(Lin et al., 2004;
White et al., 1994).
Inhibition of PR apoptosis reveals that many dorsal PRs adopt a DRA fate in
the Apc1 mutant adult, as indicated by both ectopic Rh3
expression and increased rhabdomere diameter
(Fig. 3). In wild-type flies,
Rh3 is present not only in both R7 and R8 of all DRA ommatidia, but also in
30% of R7 cells in pale ommatidia that are randomly distributed throughout the
retina (Chou et al., 1996;
Papatsenko et al., 1997)
(Fig. 3A). By contrast, in the
Apc1 mutant retina, Rh3 is found in many more inner PRs throughout
the entire dorsal half of the retina (Fig.
3B), and a corresponding increase in the rhabdomere diameter of
these inner PRs, indicative of DRA fate, is also observed
(Fig. 3C,D). Taken together,
our data support the previously proposed model for a retinal Wg gradient
(Tomlinson, 2003), and also
indicate that Apc1 loss induces both PR apoptosis in response to
high-level Arm signaling, and DRA fate specification, which is triggered by
intermediate-level Arm signaling.
Intermediate-level Arm signaling is sufficient to induce ectopic homothorax expression in a fraction of ventral ommatidia
In the wild-type pupal retina, Hth is restricted primarily to the dorsal
eye (Fig. 2A,B) by the dorsal
selector genes araucan (ara), caupolican and
mirror, which encode homologous homeodomain transcription factors
that form the Iroquois complex (IRO-C)
(Gomez-Skarmeta et al., 1996;
Wernet et al., 2003).
Therefore, our expectation was that, in the Apc1 mutant, ectopic
hth expression would also be confined to the dorsal retina. However,
we find that ectopic hth expression is not only induced throughout
the dorsal half of the retina, but is also present in a fraction of ventral
PRs in the Apc1Q8 mutant
(Fig. 4A,B).
Thus we sought to determine whether in the Apc1 mutant, ectopic
Arm signaling partially overrides the dorsal restriction of hth
expression by IRO-C. Ectopic expression of any one member of the IRO-C complex
in the ventral ommatidia of wild-type flies induces a large increase the
number of PRs at the ventral periphery that express hth, resulting in
the formation of an ectopic `ventral rim area', containing one to three rows
of hth-expressing ommatidia
(Tomlinson, 2003;
Wernet et al., 2003)
(Fig. 4A,C). We expressed
ara ectopically in all Apc1 mutant PRs using an eye-specific
GMR promoter, and found that, in the presence of Ara, all ommatidia
in the ventral Apc1 mutant retina express hth, although not
always in both R7 and R8 (Fig.
4D). Thus, although Apc1 loss induces hth expression in
both the dorsal and ventral retina, this expression is restricted primarily to
the dorsal half by the IRO-C proteins.
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) under
control of a GMR promoter. In this genotype, no PR apoptosis is
observed, indicating that high-level Arm signaling is not induced; however,
intermediate-level Arm signaling in these GMR>armS10 retinas is
sufficient to induce hth expression not only in nearly all dorsal
ommatidia, but also in a fraction of ventral ommatidia
(Fig. 4G,H). To determine
whether higher levels of Arm signaling would induce hth expression in
all ventral ommatidia, we expressed full-length Arm in all photoreceptors
under the control of an elav promoter, which drives strong expression
in all neurons. In this genotype, nearly all PRs undergo apoptosis, indicating
that high-level Arm signaling is induced throughout the retina (data not
shown). We find that in elav>arm flies, hth expression
not only expands to encompass the entire dorsal half of the retina, but also
is present in an increased number of ventral ommatidia
(Fig. 4E,F). However, despite
the presence of high-level Arm signaling in all PRs, hth expression
is found only in a fraction of ventral ommatidia, as is also observed in the
Apc1 mutant (Fig. 4B).
We conclude that in a subset of ventral ommatidia, intermediate-level Arm
signaling in PRs is sufficient to override the requirement for IRO-C in
inducing hth expression. However, neither intermediate nor high-level
Arm signaling can completely override this IRO-C requirement in all ventral
ommatidia.
Intermediate-level Arm signaling induces a shortened photoreceptor length
In the wild-type retina, photoreceptors extend the entire length of the
ommatidium, tapering slightly in diameter from the lens to the base
(Wolff and Ready, 1993)
(Fig. 5A-C). Inhibition of
apoptosis in Apc1 mutants, either by expression of the caspase
inhibitor p35, or by elimination of the cell death effectors reaper,
grim and hid, reveals that the majority of PRs are present, but
that these surviving PRs are markedly shorter
(Ahmed et al., 1998)
(Fig. 5D,E). By comparison to
wild-type PRs, the surviving Apc1 mutant PRs have slightly reduced
diameters at the very apical surface of the retina; at more basal levels, PRs
are either not detectable or the PR diameter is markedly diminished. Reducing
the gene dosage of arm by only one-half suppresses this shortening of
PR length, indicating that elevated Arm levels induce the shortened PR
morphology (Ahmed et al.,
1998).
To determine whether Arm-mediated signaling results in shortened PRs, we
used a GMR promoter to express the activated ArmS10
protein ubiquitously in otherwise wild-type retinas. As noted above, no PR
apoptosis is observed in this genotype, indicating a lack of high-level Arm
signaling; however, we find that intermediate-level Arm signaling in
GMR>armS10 retinas induces a shortening of PR length, resulting in
a morphological appearance that is identical to that induced by Apc1
loss (Ahmed et al., 1998)
(Fig. 5F-H). These data suggest
that although high-level Arm signaling induces apoptosis, intermediate-level
Arm signaling induces both a DRA fate and a shortening of PR length.
Arm functions not only as a transcriptional activator, but also as an
essential component in the formation and maintenance of cadherin-based
adherens junctions (Cox et al.,
1996; Muller and Wieschaus,
1996). To determine which of these functions results in shortened
PR length, we analyzed the requirement for Arm's co-transcriptional activator
dTCF/Pangolin (Pan) (Brunner et al.,
1997; van de Wetering et al.,
1997). In Apc1Q8 mutant flies that are also
heterozygous for the pan13 null allele
(Brunner et al., 1997), cell
death is partially suppressed, but a severe defect in PR length persists;
shortened PRs are observed in all ommatidia
(Fig. 6A,B)
(Ahmed et al., 1998). However,
a further reduction in dTCF activity, as is present in flies homozygous for
the hypomorphic allele panER1
(Brunner et al., 1997),
partially suppresses not only apoptosis, but also the shortened PR length in
the Apc1 mutant (Fig.
6C,D). These data indicate that the shortened PR length requires
both Arm and dTCF activity, and therefore is likely to involve
Arm/dTCF-mediated transcription.
|
We sought to determine whether we could distinguish the levels of Arm
signaling that induce DRA fate specification from those that induce shortening
of PR length. We found that although reducing the arm gene dosage by
only one-half in the Apc1 mutant was sufficient to partially suppress
both the cell death and the shortened PR length
(Ahmed et al., 1998), there was
no suppression of ectopic hth expression (data not shown). Together,
these results indicate that three distinct responses to Arm signaling are
specified by three distinct levels of Arm activity: PR apoptosis is induced by
the highest levels of Arm signaling, shortened PR length is induced by lower
levels, and even lower levels are sufficient to induce hth
expression.
Reduction in Apc activity by less than twofold decreases the effective threshold at which Wg elicits a cellular response
To test the model that Apc is present in vast excess, we examined the
effects of relatively modest reductions in Apc activity on Arm signaling. The
two Drosophila Apc proteins have largely redundant functions, such
that, in most cell types, the complete loss of either Apc protein singly has
no functional consequence on Wg-dependent patterning
(Ahmed et al., 2002;
Akong et al., 2002a). One
exception is in retinal photoreceptors, in which both Apc proteins are
required to negatively regulate Arm, but the relative contribution of Apc1
towards total Apc activity is much greater than that of Apc2. Although both
Apc proteins are expressed in PRs (see Fig. S3 in the supplementary material),
Apc2 levels are low enough that inactivation of Apc1 is sufficient to induce
ectopic, high-level Arm signaling (Ahmed et
al., 1998; Ahmed et al.,
2002) (Fig. 1).
Overexpression of Apc2 can compensate for Apc1 loss, revealing that even in
PRs, the two Apc proteins are functionally equivalent
(Ahmed et al., 2002). Thus, the
reduction of Apc2 levels in photoreceptors provides an opportunity to
determine the effects of a less than twofold reduction in total Apc activity
on Arm signaling.
We examined the three concentration-dependent PR responses to Arm signaling
in homozygous Apc2 mutants that contain a strong hypomorphic allele,
Apc233 (see Fig. S4 in the supplementary material). In
Apc233 mutants, we find no increase in the number of PRs
that either undergo apoptosis or have a shortened length when compared with
wild type, the two cellular responses that require higher levels of Arm
signaling (data not shown). By contrast, we find that more photoreceptors
express hth in Apc233 mutants
(Fig. 7). Strikingly, in the
Apc2 mutant, ectopic hth expression occurs only in PRs that
are immediately adjacent to those that normally express hth,
resulting in a wider zone of hth expression. Specifically, in
wild-type flies, hth expression is restricted to the most peripheral
ommatidia in the dorsal half of the retina, and also to a small fraction of
ommatidia in the `second row' and `third row', which are immediately adjacent
to the outermost row (Wernet et al.,
2003) (Fig. 7A,
arrowheads). By contrast, in homozygous Apc233 mutants,
approximately twice as many ommatidia in the second row express hth
(Fig. 7B,E). In addition, 82%
of Apc2 mutant retinas (n=51), but only 28% of wild-type
retinas (n=37), contain at least one ommatidium in the third row that
expresses hth. The increased number of ommatidia in the second and
third row of the Apc2 mutant retina that ectopically express
hth is highly significant (P<10-15;
Fig. 7E). We find similar
results in pupae that are transheterozygous for the Apc233
allele and a chromosomal deficiency that eliminates the entire Apc2
gene, Df(3R)w6 (Fig.
7C,E). In addition, the introduction of two copies of an
Apc2 transgene (Ahmed et al.,
2002) into homozygous Apc233 mutants reduces
the number of second and third row ommatidia expressing hth to
wild-type levels, while having no effect on hth expression in
wild-type flies (Fig. 7D,E; see
Fig. S5 in the supplementary material). Together, these data rule out the
possibility that the ectopic hth results from background mutations on
the Apc233 chromosome. These results indicate that
reducing total Apc activity by less than twofold can shift the threshold for
response to Wg.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Akong et al., 2002a).
Simultaneous inactivation of both Apc proteins results in ectopic Arm
signaling in nearly all, if not all, cells, indicating that Apc is required to
prevent Arm signaling in the absence of Wg stimulation. In contrast with the
prevailing model for Wg transduction, which proposes that Apc is present in
vast excess, the work presented here reveals that a less than twofold
reduction in Apc activity can shift the threshold for the response to Wg. We
conclude that by negatively regulating Arm, Apc prevents ectopic Arm activity
not only where Wg is absent, but also within the range of the Wg gradient. Translation of a gradient of Wg morphogen activity to quantitatively distinct levels of Arm signaling is required to induce concentration-dependent cellular responses, although the mechanisms by which this occurs remain uncertain. Our results reveal that in regions of low Wg concentration, reducing total Apc activity by less than twofold results in aberrant cell fate specification (Fig. 7G). A morphogen model predicts that the low Wg concentration present in this region of the gradient is below the threshold necessary to trigger a detectable cellular response. We find that this is the only region within the Wg gradient where a relatively small reduction in total Apc activity elicits an ectopic cellular response, and this response is characteristic of intermediate-level Arm signaling. Thus, our results reveal that Apc activity is in excess in regions where Wg is absent, but is not in vast excess within the range of the Wg gradient. Together, our data indicate that Apc activity is present near the minimal level required to prevent ectopic Arm signaling and thereby ensure accurate graded responses.
In Xenopus egg extracts, the levels of Axin are several magnitudes
lower than the levels of other proteins in the destruction complex, suggesting
that only Axin is a limiting component in Arm proteolysis, whereas Apc is
present in vast excess (Lee et al.,
2003; Salic et al.,
2000). How can these biochemical data be reconciled with our in
vivo data, which indicate that Apc is not present in excess within the range
of the Wg gradient? One possibility is that the levels of Apc in
Xenopus eggs are much greater than those present in
Drosophila photoreceptors. Alternatively, total Apc levels could be
present in excess regardless of cell type or organism, but the relevant pool
contributing to destruction complex activity, distinguished by either
post-translational modification and/or intracellular localization, might be
present near threshold levels. A correlation between the degree of reduction
in the activity of the fly and mammalian Apc proteins with the level of
β-catenin/Arm signaling has been demonstrated in several other
developmental contexts and in tumorigenesis
(Ahmed et al., 2002;
Akong et al., 2002a;
Akong et al., 2002b;
Benhamouche et al., 2006;
Hayden et al., 2007;
Kielman et al., 2002;
McCartney et al., 2006;
Smits et al., 1999). Thus data
from diverse experimental models indicate that the level of Apc contributes to
the level of β-catenin/Arm signaling.
How is a gradient of Wg concentration translated into quantitatively
distinct levels of Arm activity? Upon Wg stimulation, inactivation of the
Axin/Zw3/Apc destruction complex is the primary event that triggers Arm
signaling (Peifer et al.,
1994; Siegfried et al.,
1994; Tolwinski et al.,
2003). Inactivation of Axin is important for downstream signal
transduction in response to Wg stimulation, and is likely to be mediated by
the translocation of Axin to the plasma membrane, and/or the degradation of
Axin (Cliffe et al., 2003;
Tamai et al., 2004;
Tolwinski et al., 2003). Thus
the local Axin concentration is likely to have a significant role in
determining whether the destruction complex is assembled, and consequently is
important in regulating Arm stability. Our findings provide in vivo evidence
that the level of destruction complex activity is crucial for accurate
patterning in response to Wg, and is dependent not only on Axin, but also on
the maintenance of Apc activity above a minimal level. We conclude that within
the range of the Wg gradient, both Axin and Apc are present near threshold
levels, and that, together, they achieve the precise levels of destruction
complex activity required for accurate graded responses.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/5/963/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Abu-Shaar, M., Ryoo, H. D. and Mann, R. S.
(1999). Control of the nuclear localization of Extradenticle by
competing nuclear import and export signals. Genes
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Ahmed, Y., Hayashi, S., Levine, A. and Wieschaus, E. (1998). Regulation of armadillo by a Drosophila APC inhibits neuronal apoptosis during retinal development. Cell 93,1171 -1182.[CrossRef][Medline]
Ahmed, Y., Nouri, A. and Wieschaus, E. (2002).
Drosophila Apc1 and Apc2 regulate Wingless transduction throughout
development. Development
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