scispace - formally typeset

Journal ArticleDOI

Negative regulation of Armadillo, a Wingless effector in Drosophila

01 Jun 1997-Development (The Company of Biologists Ltd)-Vol. 124, Iss: 11, pp 2255-2266

TL;DR: It is shown that Armadillo(S10) has escaped from negative regulation by Zeste white-3 kinase, and thus accumulates outside junctions even in the absence of Wingless signal, suggesting that it is less rapidly targeted for degradation.
Abstract: Drosophila Armadillo and its vertebrate homolog beta-catenin play essential roles both in the transduction of Wingless/Wnt cell-cell signals and in the function of cell-cell adherens junctions. Wingless and Wnts direct numerous cell fate choices during development. We generated a mutant protein, Armadillo(S10), with a 54 amino acid deletion in its N-terminal domain. This mutant is constitutively active in Wingless signaling; its activity is independent of both Wingless signal and endogenous wild-type Armadillo. Armadillo's role in signal transduction is normally negatively regulated by Zeste-white 3 kinase, which modulates Armadillo protein stability. Armadillo(S10) is more stable than wild-type Armadillo, suggesting that it is less rapidly targeted for degradation. We show that Armadillo(S10) has escaped from negative regulation by Zeste white-3 kinase, and thus accumulates outside junctions even in the absence of Wingless signal. Finally, we present data implicating kinases in addition to Zeste white-3 in Armadillo phosphorylation. We discuss two models for the negative regulation of Armadillo in normal development and discuss how escape from this regulation contributes to tumorigenesis.
Topics: Armadillo (67%)

Summary (2 min read)

INTRODUCTION

  • During embryonic development, cells acquire information from many sources about the fates they should choose.
  • In their current model, soluble Armadillo (or its vertebrate homolog β-catenin) is rapidly degraded in the absence of Wg/Wnt signal, and thus its steady state level outside adherens junctions is low (Peifer et al., 1994a; van Leeuwen et al., 1994).
  • The next step remained mysterious until recently.
  • The authors and others recently extended this work in Drosophila, showing that the fly TCF family member dTCF plays an essential role in Wingless signaling in vivo (van de Wetering et al., 1997; Brunner et al., 1997).
  • In the experiments described here, the authors focused on one aspect of the signaling pathway, namely the means by which Zw3 negatively regulates Arm via effects on Arm stability.

MATERIALS AND METHODS

  • Armadillo mutants and other fly stocks Mutations were introduced into a c-myc-tagged arm cDNA (Orsulic and Peifer, 1996b; details upon request).
  • ArmS10 (a.a. 34-87 deleted) was created using an in vitro mutagenesis kit (USB) as recommended by the manufacturer.
  • Several homozygous lines of each construct were established; mutant protein expression was tested by immunoblotting.
  • ArmYD35, armH8.6 and armXP33 are described by Peifer and Wieschaus (1990), zw3M11-1 is described by Siegfried et al. (1994), and wgIG22 is described by Nüsslein-Volhard and Wieschaus (1980).
  • Most GAL4 stocks were from the Bloomington Drosophila stock center; en-GAL4 was provided by A. Brand.

Genetic tests of function

  • The authors performed four genetic tests of armS10 at 25°C, using a line that expresses mutant protein at approximately wild-type levels.
  • First, the authors tested armS10 in a zygotic null arm background, by crossing armYD35/FM7; e22c-GAL4/+ females to armS10 homozygous males.
  • Cuticles were prepared as described by Wieschaus and Nüsslein-Volhard (1986).
  • Con ASepharose fractionation using NET buffer was as described by Pai et al. (1996).
  • (1) anti-myc antibody; as in Orsulic and Peifer (1996b), (2) anti-Arm; as in Peifer et al. (1991), (3) anti-Engrailed and Anti-Wg; as in DiNardo et al. (1985), also known as Protein localization in situ.

RESULTS

  • Sequences in Armadillo’s N-terminal domain negatively regulate Armadillo activity in Wingless signaling When activated by e22c-GAL4, ArmS10, Arm∆N and ArmS2 all accumulate to levels roughly similar to that of wild-type Arm (Fig. 1B,C; data not shown); when corrected for gene copy number ArmS10 levels are approx.
  • Expression of ArmS10 in such armH8.6 mutant embryos dramatically alters their phenotype; the embryos secrete only naked cuticle (Table 1; Fig. 4H), and overlap in phenotype with wild-type embryos expressing ArmS10.
  • A low level of both proteins remained even at the longest timepoint; this pool of long-lived protein may be junctional.
  • Thus Zw3 kinase does not regulate the stability of ArmS10 outside junctions; in fact ArmS10 behaves in a wild-type background in a fashion very similar to wild-type Arm in a zw3 mutant background, consistent with the idea that ArmS10 has escaped from regulation by Zw3.

A region of Armadillo negatively regulates Armadillo’s activity in Wg signal transduction

  • The authors identified a region of Arm essential for this negative regulation.
  • The authors confirmed this by showing that the activity of ArmS10 is independent of Wg signal, as assayed both by its effects on embryonic pattern and its ability to activate the downstream genes engrailed and wg.
  • Alternately, these mutants could both evade degradation and act in signaling themselves.
  • These data suggested a simple model, whereby Zw3/GSK kinase negatively regulates Arm/β-catenin by directly phosphorylating it on a site within the N terminus.
  • In support of this alternative model, the authors found that their earlier data documenting differences in Arm phosphorylation between wild-type and zw3 mutants are at least in part an indirect result of alterations of the ratio of junctional to soluble Arm.

Did you find this useful? Give us your feedback

Content maybe subject to copyright    Report

INTRODUCTION
During embryonic development, cells acquire information
from many sources about the fates they should choose. One
key source of information is communication among neighbor-
ing cells, which induces new cell fates and maintains old ones.
The Wingless/Wnt family of cell-cell signaling proteins directs
numerous processes, including anterior-posterior patterning of
the
Drosophila embryonic epidermis, dorsal-ventral patterning
in early vertebrate embryos, and dorsal-ventral patterning of
the limbs of flies and vertebrates (reviewed by Parr and
McMahon, 1994).
Components of the Drosophila Wingless (Wg) signal trans-
duction pathway have been identified; similar proteins also
transduce certain vertebrate Wnt signals (reviewed by Orsulic
and Peifer, 1996a). One component, Armadillo (Arm), accu-
mulates both in adherens junctions, where it regulates cell
adhesion (Cox et al., 1996), and in the cytoplasm and nucleus
of certain cells, where it mediates Wg signal transduction. In
our current model, soluble Armadillo (or its vertebrate
homolog
β-catenin) is rapidly degraded in the absence of
Wg/Wnt signal, and thus its steady state level outside adherens
junctions is low (Peifer et al., 1994a; van Leeuwen et al., 1994).
This degradation requires the action of both the
serine/threonine (Ser/Thr) kinase Zeste white-3 (Zw3; its ver-
tebrate homolog is glycogen synthase kinase 3-β (GSK); Peifer
et al., 1994a; Siegfried et al., 1994) and the product of the
tumor suppressor protein adenomatous polyposis coli (APC;
Munemitsu et al., 1995; Rubinfeld et al., 1996). In contrast,
Wg interaction with its receptor (Bhanot et al., 1996) activates
Dishevelled (Yanagawa et al., 1995), which counteracts Zw3
activity (Cook et al., 1996). This stabilizes Arm in the
cytoplasm and nucleus (Orsulic and Peifer, 1996b).
The next step remained mysterious until recently. The first
clue came from the realization that vertebrate β-catenin can
form a complex with DNA-binding proteins of the TCF/LEF
family, and these proteins could, when ectopically expressed
in Xenopus, alter dorsal-ventral patterning in a way which
suggested that they play a role in Wnt signaling (Behrens et
al., 1996; Molenaar et al., 1996; Huber et al., 1996). We and
others recently extended this work in Drosophila, showing that
the fly TCF family member dTCF plays an essential role in
Wingless signaling in vivo (van de Wetering et al., 1997;
Brunner et al., 1997). The active transcription factor is a
complex between Arm and dTCF, and this complex directly
regulates the expression of Wingless-responsive genes (van de
Wetering et al., 1997; Riese et al., 1997).
In the experiments described here, we focused on one aspect
of the signaling pathway, namely the means by which Zw3
negatively regulates Arm via effects on Arm stability. Since
Zw3 is a Ser/Thr kinase, Arm degradation may be regulated by
phosphorylation. Two possible Zw3 targets have been identi-
fied. One is Arm itself. Hypophosphorylated Arm accumulates
in zw3 mutants (Peifer et al., 1994b). A Zw3/GSK phosphory-
2255
Development 124, 2255-2266 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
DEV5115
Drosophila Armadillo and its vertebrate homolog
ββ
-catenin
play essential roles both in the transduction of
Wingless/Wnt cell-cell signals and in the function of cell-
cell adherens junctions. Wingless and Wnts direct
numerous cell fate choices during development. We
generated a mutant protein, Armadillo
S10
, with a 54 amino
acid deletion in its N-terminal domain. This mutant is con-
stitutively active in Wingless signaling; its activity is inde-
pendent of both Wingless signal and endogenous wild-type
Armadillo. Armadillo’s role in signal transduction is
normally negatively regulated by Zeste-white 3 kinase,
which modulates Armadillo protein stability. Armadillo
S10
is more stable than wild-type Armadillo, suggesting that it
is less rapidly targeted for degradation. We show that
Armadillo
S10
has escaped from negative regulation by
Zeste white-3 kinase, and thus accumulates outside
junctions even in the absence of Wingless signal. Finally,
we present data implicating kinases in addition to Zeste
white-3 in Armadillo phosphorylation. We discuss two
models for the negative regulation of Armadillo in normal
development and discuss how escape from this regulation
contributes to tumorigenesis.
Key words: Armadillo, β-catenin, Wingless, Wnt, Zeste white-3,
Drosophila
SUMMARY
Negative regulation of Armadillo, a Wingless effector in
Drosophila
Li-Mei Pai
1
, Sandra Orsulic
1,
*, Amy Bejsovec
2
and Mark Peifer
1,†
1
Department of Biology and Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599-
3280, USA
2
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston IL 60208, USA
*Present address: Max-Planck Institüt für Immunbiologie, D-79108 Freiberg, Germany
Corresponding author (e-mail: peifer@unc.edu)

2256
lation consensus site is conserved between Arm and β-catenin
(Peifer et al., 1994b); GSK phosphorylates β-catenin in vitro,
using this conserved site (Yost et al., 1996). Another possible
target of Zw3/GSK is APC; GSK phosphorylates APC,
promoting binding of APC to β-catenin and ultimately β-
catenin degradation (Rubinfeld et al., 1996; Munemitsu et al.,
1995). Regulation of Arm/β-catenin degradation not only plays
a key role in Wg/Wnt signaling, but also is a target for activa-
tion in both colon cancer and melanoma (Korinek et al., 1997;
Morin et al., 1997; Rubinfeld et al., 1997).
Here we report that deletion of the GSK/Zw3 consensus
phosphorylation site in the N-terminal domain of Arm relieves
the normal negative regulation by Zw3, constitutively activat-
ing Arm in Wg signal transduction. This mutant Arm no longer
depends on Wg signal nor on endogenous wild-type Arm for
activity, and thus it not only escapes negative regulation but
also can itself signal. Mutant protein accumulates at high levels
in the cytoplasm and nucleus of all cells, regardless of whether
they receive Wg, mirroring the accumulation of wild-type Arm
in a zw3 mutant. Consistent with this, mutations in zw3 do not
increase the stability of the mutant Arm. The pool of Arm
protein in adherens junctions remains fully phosphorylated in
a zw3 mutant, suggesting that alternate kinases can phospho-
rylate Arm and raising the possibility that Zw3 may affect Arm
phosphorylation indirectly by altering its stability. We discuss
two alternative roles for Zw3 in Arm regulation.
MATERIALS AND METHODS
armadillo
mutants and other fly stocks
Mutations were introduced into a c-myc-tagged arm cDNA (Orsulic
and Peifer, 1996b; details upon request). arm
S10
(a.a. 34-87 deleted)
was created using an in vitro mutagenesis kit (USB) as recommended
by the manufacturer. Wild-type or mutant cDNAs were introduced
into the pUAST vector (Brand and Perrimon, 1993). arm
N
(a.a. 1-
128 deleted) is described by Pai et al. (1996). y arm
+
w ies were used
for transformation. Several homozygous lines of each construct were
established; mutant protein expression was tested by immunoblotting.
arm
YD35
, arm
H8.6
and arm
XP33
are described by Peifer and Wieschaus
(1990), zw3
M11-1
is described by Siegfried et al. (1994), and wg
IG22
is described by Nüsslein-Volhard and Wieschaus (1980). Germline
clones were produced as described by Peifer et al. (1994a). Most
GAL4 stocks were from the Bloomington Drosophila stock center;
en-GAL4 was provided by A. Brand.
Genetic tests of function
We performed four genetic tests of arm
S10
at 25°C, using a line that
expresses mutant protein at approximately wild-type levels. Progeny
were examined as follows. Eggs were collected and hatch rates deter-
mined, cuticles of hatched larvae and unhatched embryos were
prepared, and adult progeny were scored for viability. First, we tested
arm
S10
in a zygotic null arm background, by crossing arm
YD35
/FM7;
e22c-GAL4/+ females to arm
S10
homozygous males. Second and
third, we tested arm
S10
in embryos with a maternal and zygotic con-
tribution composed entirely of arm
XP33
or arm
H8.6
mutant protein.
Germline clones of arm
XP33
or arm
H8.6
were generated as described
by Peifer et al. (1994a). Females carrying germline clones and het-
erozygous for e22c-GAL4 were mated to arm
S10
homozygous males.
Fourth, we tested arm
S10
in a wg background, by analyzing progeny
of the recombinant stock arm
S10
wg
IG22
/CyO. Cuticles were prepared
as described by Wieschaus and Nüsslein-Volhard (1986).
Biochemical and cell biological studies
We used monoclonal anti-c-myc (purified from 9E10 cell supernatant;
Orsulic and Peifer, 1996b; 1:200 for immunofluorescence and un-
purified culture supernatant for immunoblotting), polyclonal anti-Arm
N2 (Peifer et al., 1994a; 1:200), polyclonal anti-Arm CT (1:40), mon-
oclonal anti-Arm 7A1 (Peifer et al., 1994a; 1:500), monoclonal anti-
Engrailed (DiNardo et al., 1985; 1:50) and polyclonal anti-Wg (van
den Heuvel et al., 1989; 1:500). Immunoprecipitation, immunoblot-
ting and cell fractionation were as in Peifer (1993). When
immunoblotting, proteins were detected either by ECL (Amersham)
or by using alkaline phosphatase-coupled secondary Ab, NBT and
BCIP (Promega), as recommended by the manufacturers. Con A-
Sepharose fractionation using NET buffer was as described by Pai et
al. (1996). Okadaic acid and PP-2A treatment were as described by
Peifer et al. (1994b). Protein localization in situ: (1) anti-myc
antibody; as in Orsulic and Peifer (1996b), (2) anti-Arm; as in Peifer
et al. (1991), (3) anti-Engrailed and Anti-Wg; as in DiNardo et al.
(1985).
RESULTS
Sequences in Armadillo’s N-terminal domain
negatively regulate Armadillo activity in Wingless
signaling
We previously characterized a number of arm mutant trans-
genes under the control of the arm promoter, and thus
expressed ubiquitously at high levels (Orsulic and Peifer,
1996b). During those experiments we generated a mutation,
arm
S10
, in which 54 amino acids of the N-terminal domain
were deleted (a.a. 34-87; Fig. 1A). Despite repeated efforts, we
were unable to obtain transformants expressing arm
S10
; in
parallel experiments multiple transformants were obtained for
all other mutants. This suggested arm
S10
is dominant lethal.
The region deleted in arm
S10
includes two interesting sequence
motifs (Fig. 1A). One is a consensus GSK/Zw3 phosphoryla-
tion site (Peifer et al., 1994b). The other is a sequence
conserved between IκB and its fly homolog cactus, surround-
ing the serines whose phosphorylation is thought to regulate
IκB ubiquitination and thus control its stability (reviewed by
Hochstrasser, 1996).
To test whether arm
S10
is dominant lethal, we expressed an
arm
S10
transgene using the inducible GAL-UAS system (Brand
and Perrimon, 1993). arm
S10
was cloned downstream of UAS
elements regulated by yeast GAL4 and introduced into flies,
where it is inactive. arm
S10
can be activated in particular tissues
at specific times by crossing arm
S10
lines to lines expressing
GAL4 in specific temporal and spatial patterns. We obtained
transgenic animals in which either arm
S10
, or another mutant,
arm
N
(lacking the entire N-terminal domain; Fig. 1A) are
under GAL4 regulation. As a control, we placed myc-tagged
wild-type Arm under GAL4 control (arm
S2
). We then used
e22c-GAL4 (which expresses GAL4 ubiquitously) to drive
essentially ubiquitous expression of our mutant Arm beginning
late in embryonic stage 9.
When activated by e22c-GAL4, Arm
S10
, Arm
N
and Arm
S2
all accumulate to levels roughly similar to that of wild-type
Arm (Fig. 1B,C; data not shown); when corrected for gene
copy number Arm
S10
levels are approx. 2.4 times those of wild-
type Arm (Fig. 1C), while Arm
N
levels are approx. 1.5 times
wild-type Arm (Arm
N#7
). When analyzed by SDS-PAGE,
L.-M. Pai and others

2257Negative regulation of Armadillo
wild-type Arm accumulates as a set of Ser/Thr phosphoryla-
tion variants (105-110×10
3
M
r
; Peifer et al., 1994b); the most
highly phosphorylated isoforms migrate most slowly (Fig. 1B).
Both Arm
S10
and Arm
N
also have multiple isoforms which
result at least in part from Ser/Thr phosphorylation (Fig. 1B).
Treatment of Arm
S10
with protein phosphatase 2A, a Ser/Thr-
specific phosphatase, eliminates all but the fastest migrating
isoform (Fig. 1D), while treatment with okadaic acid, an
inhibitor of Ser/Thr-phosphatases, increases the levels of
slower migrating Arm
S10
and Arm
N
isoforms (Fig. 1E).
Most of the wild-type Arm in an embryo is in adherens
junctions, where it is highly phosphorylated; there is relatively
little soluble Arm, which is less highly phosphorylated (Peifer
et al., 1994b; Fig. 1B). In contrast, the least highly phos-
phorylated isoforms of Arm
S10
predominate (Fig. 1B), resem-
bling the pattern of accumulation of wild-type Arm in zw3
mutants (Peifer et al., 1994b). This is not due to ectopic
expression; wild-type Arm expressed under GAL4 control is
normally phosphorylated (data not shown).
In wild-type embryos, one can follow cell fate choices using
cuticular markers; anterior cells within each segment secrete
cuticle covered with denticles while posterior cells secrete
naked cuticle (Fig. 2A,F). This pattern depends on Wg. A
single row of cells underlying the future naked cuticle secrete
Wg and their neighbors respond to this by choosing posterior
fates. In embryos mutant for either wg signal or for arm, which
is required for signal transduction, all cells assume anterior
fates and secrete denticles (Nüsslein-Volhard and Wieschaus,
1980). In the presence of high and ubiquitous levels of Wg, all
cells assume posterior fates and secrete naked cuticle (Noor-
dermeer et al., 1992; Sampedro et al., 1993; Yoffe et al., 1995).
Ubiquitous expression of either amino-terminally deleted
Arm mutant, Arm
S10
or Arm
N
, mimics the effects of ubiqui-
tous Wg expression, producing a phenotype opposite to that of
an arm mutant (Fig. 2). arm
S10
lines produce embryos with
only naked cuticle or a few denticles (Fig. D,E,G), a phenotype
similar to that caused by very high levels of ubiquitous Wg
(Yoffe et al., 1995). Expression of arm
S10
using a second ubiq-
uitously expressed driver, 69B-GAL4, had similar effects (Fig.
2). arm
N
lines display partial ablation of the denticle belts,
particularly along the ventral midline (Fig. 2B,C); this
phenotype overlaps that of arm
S10
, but is somewhat weaker and
more variable among different transformant lines. The arm
N
phenotype resembles the phenotype seen when Wg is ubiqui-
tously expressed at lower levels (Noordermeer et al., 1992). As
a control we expressed wild-type Arm (arm
S2
; Fig. 1A) ubiq-
uitously, using e22c- or 69B-GAL4. This had no phenotypic
consequences; in fact animals carrying Arm
S2
driven by e22c-
GAL4 survived to adulthood (data not shown). Further, ubiq-
Fig. 1. Arm
S10
and Arm
N
expression and phosphorylation.
(A) Mutant proteins used in this study. Note that Arm
S2
and Arm
S10
are tagged with the myc epitope while Arm
N
is not tagged. The
region deleted in Arm
S10
includes a match to the GSK-3β consensus
phosphorylation site (Peifer et al., 1994b). It also contains a match to
a region conserved between IκB and its fly homolog Cactus,
surrounding the serines whose phosphorylation is thought to regulate
IκB ubiquitination and thus stability. (B) Both Arm
S10
and Arm
N
have multiple isoforms. Protein from 0-16 hour old embryos
expressing Arm
S10
or Arm
N
was analyzed by immunoblotting with
anti-ArmCT (recognizing wild-type, Arm
S10
and Arm
N
) or anti-c-
myc (recognizing Arm
S10
). (C) A similar experiment, using ECL
detection to allow quantitation; BicD was used to control loading.
(D,E) Arm
S10
is phosphorylated on serine/threonine.
(D) Immunoprecipitates using anti-ArmCT from embryos expressing
Arm
S10
were treated with PP-2A or PP-2A plus its inhibitor okadaic
acid. (E) Isolated embryonic cells from Arm
S10
or Arm
N
embryos
were incubated in D-22 medium with or without the phosphatase
inhibitor okadaic acid.

2258
uitous expression of wild-type Arm using e22c-GAL4 rescued
the embryonic lethality and cuticular phenotype of the zygotic
null arm
YD35
(data not shown).
To confirm that the phenotype was caused by the mutant
proteins, and to begin to investigate whether their effects are
cell autonomous, we expressed them in more restricted
patterns. We utilized an engrailed-GAL4 (A. Brand and K.
Yoffe, personal communication), which drives transgene
expression in posterior cells of each segment; these include
both the most posterior cells secreting naked cuticle and the
cells secreting the anteriormost row of denticles. When arm
S10
is expressed in the engrailed domain, the first row of denticles
and only the first row of denticles is replaced by naked cuticle
(Fig. 2H versus I), suggesting that Arm
S10
can act cell-
autonomously. We also drove arm
N
with hairy-GAL4, which
directs expression in even-numbered segments at the cellular
blastoderm stage; the resulting embryos had fewer denticles in
many even-numbered segments (data not shown).
Both arm
S10
and arm
N
alter the expression of two genes that
are known targets of Wg
signaling: engrailed, expressed
in two rows of cells immediately
posterior to the wg-expressing
cells (Fig. 3A), and wg itself (Fig.
3G). Ubiquitous expression of
either arm
S10
(Fig. 3B) or arm
N
(data not shown) causes a
posterior expansion in the
domain of en-expressing cells
and induces an ectopic stripe of
the endogenous Wg (Fig. 3H), as
does ubiquitous Wg expression
(Noordermeer et al., 1992; Yoffe
et al., 1995).
In a variety of different assays,
arm
N
was slightly less potent
than arm
S10
. This was a differ-
ence of degree rather than a qual-
itative difference. This may be
explained by the fact that arm
N
accumulates to slightly lower
levels than arm
S10
(Fig. 1B).
arm
N
is found as several widely
spaced isoforms. The first
methionine in arm
N
is not the
natural methionine of Arm; this
could influence its efficiency of
translation, leading to the
slightly lower levels. The
isoforms may represent starts at
other, more internal methionine
codons, or they may mean that
the protein is somewhat less
stable than normal. It is worth
noting that arm
N
possesses
activated signaling activity
despite the fact that it lacks the
α-catenin binding site (Pai et al.,
1996), consistent with indepen-
dent roles for Arm in signaling
and cell adhesion.
The activity of Arm
S10
is independent of Wingless
signal
These data suggest that arm
S10
has escaped normal negative
regulatory cues and has become constitutively active. If so, it
should be independent of endogenous Wg. To test this we
expressed Arm
S10
in a wg mutant where no cell is exposed to
Wg. We evaluated cell fate choices by examining both cuticle
pattern and Wg target gene expression. wg null embryos are
shorter than wild-type and secrete only denticles with no naked
cuticle (Nüsslein-Volhard and Wieschaus, 1980; Fig. 4C).
Expression of Arm
S10
in a wg mutant dramatically alters this
(Fig. 4D), producing a cuticle phenotype similar to that of
embryos expressing Arm
S10
in a wild-type background (Fig.
4B). Thus Arm
S10
acts independently of Wg. Once again,
Arm
N
is slightly less active than Arm
S10
. wg mutant embryos
expressing Arm
N
secrete naked cuticle interspersed with
denticles on their ventral surface (data not shown), while the
dorsal surface is unchanged from the wg mutant phenotype. In
contrast, Arm
S10
expression rescues pattern elements on both
L.-M. Pai and others
Fig. 2. Ubiquitous expression of Arm
S10
and Arm
N
transforms cells to posterior cell fates. Cuticle
preparations of wild-type embryos (A,F,H), and of embryos expressing either Arm
S10
(D,E,G,I) or
Arm
N
(B,C) under GAL4 control. e22c-GAL4 and 69B-GAL4 drive essentially ubiquitous
expression, while en-GAL4 drives expression specifically in the posterior compartment.
(B-G) Ubiquitous expression of either Arm
S10
or Arm
N
using e22c-GAL4 drove cells into naked
cuticle fates; Arm
N
lines were weaker in their effects than Arm
S10
lines. The weakest Arm
N
lines
only lack a few denticles near the ventral midline (data not shown). (B) In stronger lines (Arm
N#6)
,
larger regions of naked cuticle appear along the midline (arrowhead). (C) In the strongest Arm
N
lines
(Arm
N#7
), most cells choose naked cuticle fates. (D,E,G) Expression of Arm
S10
under the control of
e22c-GAL4 or 69B-GAL4 caused nearly all cells to choose naked cuticle fates, leaving only a few
denticles (arrowheads); unlike zw3 mutants, head structures remain nearly normal. (H,I) When en-
GAL4 was used to specifically express Arm
S10
in the posterior compartment, which gives rise both to
the most posterior cells of the naked cuticle and the anterior row of denticles (arrowheads), the
anterior row of denticles was specifically transformed to naked cuticle (arrowheads).

2259Negative regulation of Armadillo
the ventral and dorsal surfaces. Using similar GAL4 drivers,
ubiquitous Wg expression produces uniform naked cuticle, as
it does in the wild-type background (unpublished data).
In the absence of Wg, Engrailed (En) expression decays
between 4 and 5 hours of development (DiNardo et al., 1988;
Fig. 3F). Ubiquitous expression of either Arm
S10
(Fig. 3D) or
Arm
N
(Fig. 3E) in a wg mutant causes expansion rather than
loss of the En expression domain. Arm
N
promotes En
expansion only in a ventral domain of cells, while more dorsal
parts of the stripe decay (Fig. 3E). Arm
S10
stabilizes and
expands the entire En stripe, although the effect is slightly less
pronounced in the dorsal region (Fig. 3D). These experiments
demonstrate that the effects of Arm
S10
or Arm
N
are indepen-
dent of Wg ligand and rule out the possibility that the ectopic
Wg expression that they induce is solely responsible for their
phenotypic effects.
The activity of Arm
S10
is independent of
endogenous Armadillo
The data above can be explained in two distinct ways. Arm
S10
might have escaped normal negative regulation and thus be
constitutively active in Wg signaling. Alternately, Arm
S10
might have no signaling activity of its own, but might poison
the degradation machinery, block degradation of endogenous
Arm, and allow it to accumulate and transduce Wg signal. To
distinguish between these two possibilities, we introduced
Arm
S10
into genetic backgrounds depleted for or devoid of
wild-type Arm.
We first introduced Arm
S10
into arm
YD35
embryos, which are
zygotically arm null but possess normal levels of maternally
contributed Arm. arm
YD35
mutant embryos have a strong
segment polarity defect and defects in dorsal closure (Fig. 4E).
When Arm
S10
is expressed in arm
YD35
mutant embryos using
e22c-GAL4, the resulting embryos differentiate essentially
only naked cuticle (Table 1; Fig. 4F), similar to the phenotype
Fig. 3. Ubiquitous expression
of Arm
S10
and Arm
N
alters
gene expression in a fashion
similar to that caused by
ubiquitous Wg. Expression of
Engrailed (A-F) and Wg (G-
H) were examined in wild-
type or wg mutant embryos
with Arm
S10
or Arm
N
expressed ubiquitously using
e22c-GAL4. (A) Wild-type
embryo at stage 9. Engrailed
is expressed in two to three
rows of cells per segment
(arrowheads). (B) In contrast,
in embryos expressing
Arm
S10
, Engrailed stripes are
broadened to three to four
rows of cells (arrowheads).
(C,F) In wg mutants Engrailed
expression begins to decay
during early stage 9 (C; arrowhead) and is gone from the epidermis by stage 10 (F; remaining expression is in the nervous system). (D,E)
Expression of either Arm
S10
(D) or Arm
N
(E) in a wg mutant prevents decay of Engrailed stripes; instead stripes are broadened. (G) In wild-
type embryos, Wg is secreted by a single row of cells (arrowhead). (H) In embryos expressing Arm
S10
, an ectopic stripe of Wg is induced in
addition to the endogenous stripe (arrowheads).
Genotypes of progeny
Phenotype
Naked
Table 1. arm
S10
activity is independent of endogenous
wild-type Arm
Naked
Denticle lawn
Naked
Cross 1
Observed
×
;
arm
S10
arm
S10
arm
YD35
e22c
+
Wild-type
Wild-type
3/8
(37.5%)
3/8
(37.5%)
1/8
(12.5%)
1/8
(12.5%)
34.7%
15%
50.3%
×
;
arm
S10
arm
S10
arm
H8.6
e22c
+
arm
H8.6
glc*
arm
YD35
+
;
arm
S10
Cross 2
Naked
arm
H8.6
+
;
e22c
arm
S10
Naked
Wild-type
1/4
(25%)
1/4
(25%)
1/4
(25%)
23.4%
50.2%
26.4%
n=709
n=337
1/4
(25%)
Predicted
+ or Y
;
e22c
arm
S10
arm
YD35
+
;
arm
S10
+
+ or Y
;
arm
S10
+
arm
YD35
Y
;
arm
S10
+
arm
YD35
Y
;
e22c
arm
S10
Denticle lawn
e22c
arm
H8.6
Y
;
e22c
arm
S10
arm
H8.6
+
;
+
arm
S10
;
+
arm
S10
arm
H8.6
Y
+
+
+
*arm
H8.6
glc indicates females whose germlines are homozygous
for the arm
H8.6
mutation.

Figures (6)
Citations
More filters

Journal ArticleDOI
Ken M. Cadigan1, Roel Nusse1Institutions (1)
TL;DR: Current understanding of Wnt function and signaling mechanisms is reviewed in a comparative approach, highlighting novelty and underscoring questions that remain, and putting emphasis on the latest findings.
Abstract: Wnt proteins are now recognized as one of the major families of developmentally important signaling molecules, with mutations in Wnt genes displaying remarkable phenotypes in the mouse, Caenorhabditis elegans, and Drosophila. Among functions provided by Wnt proteins are such intriguing processes as embryonic induction, the generation of cell polarity, and the specification of cell fate. Until recently, our knowledge of the molecular mechanism of Wnt signaling was very limited, but over the past year, several major gaps have been filled. These include the identification of cell-surface receptors and a novel mechanism of relaying the signal to the cell nucleus. In addition, several components of Wnt signaling are implicated in the genesis of human cancer. These insights have come from different corners of the animal kingdom and have converged on a common pathway. At this junction in this rapidly evolving field, we review our current understanding of Wnt function and signaling mechanisms, doing so in a comparative approach. We have put emphasis on the latest findings, highlighting novelty and underscoring questions that remain. For additional literature, we refer to several previous reviews (McMahon 1992; Nusse and Varmus 1992; Klingensmith and Nusse 1994; Miller and Moon 1996; Moon et al. 1997). We have limited the number of references, particularly in the tables. Fully referenced forms of these tables can be found on the Wnt homepage (http://wwwleland.stanford.edu/∼rnusse/wntwindow.html).

2,576 citations


Cites background from "Negative regulation of Armadillo, a..."

  • ...Both in flies and Xenopus, these mutant proteins are no longer sensitive to Zw3/GSK-3 regulation (Yost et al. 1996; Pai et al. 1997)....

    [...]

  • ...This has led to a model where Wnt acts to negatively regulate the Zw3/GSK-3 kinase, though the data equally support Zw3/GSK-3 acting in parallel as a repressor of Arm/b-catenin....

    [...]

  • ...A complex among Zw3/GSK-3, Arm/b-catenin, and adenomatous polyposis coli The Arm protein is similar to vertebrate b-catenin and plakoglobin, proteins binding to E-cadherin (McCrea et al. 1991) and linking adhesion complexes to the cytoskeleton....

    [...]

  • ...Although the above results make a compelling case for the importance of the amino-terminal phosphorylation sites in regulating Arm/b-catenin stability and signaling activity, the data that Zw3/GSK-3 is the direct kinase are less convincing....

    [...]

  • ...Thus, there is precedence for the idea that Wnt proteins inhibit Zw3/GSK-3 through covalent modification....

    [...]


Journal ArticleDOI
Andreas Wodarz, Roel Nusse1Institutions (1)
TL;DR: Over the past two years the understanding of Wnt signaling has been substantially improved by the identification of Frizzled proteins as cell surface receptors for Wnts and by the finding that beta-catenin, a component downstream of the receptor, can translocate to the nucleus and function as a transcriptional activator.
Abstract: Wnt genes encode a large family of secreted, cysteine-rich proteins that play key roles as intercellular signaling molecules in development. Genetic studies in Drosophila and Caenorhabditis elegans, ectopic gene expression in Xenopus, and gene knockouts in the mouse have demonstrated the involvement of Wnts in processes as diverse as segmentation, CNS patterning, and control of asymmetric cell divisions. The transduction of Wnt signals between cells proceeds in a complex series of events including post-translational modification and secretion of Wnts, binding to transmembrane receptors, activation of cytoplasmic effectors, and, finally, transcriptional regulation of target genes. Over the past two years our understanding of Wnt signaling has been substantially improved by the identification of Frizzled proteins as cell surface receptors for Wnts and by the finding that beta-catenin, a component downstream of the receptor, can translocate to the nucleus and function as a transcriptional activator. Here we review recent data that have started to unravel the mechanisms of Wnt signaling.

1,982 citations


Journal ArticleDOI
04 May 2000-Nature
TL;DR: It is shown that the zebrafish silberblick locus encodes Wnt11 and that Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation, and that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue.
Abstract: Vertebrate gastrulation involves the specification and coordinated movement of large populations of cells that give rise to the ectodermal, mesodermal and endodermal germ layers. Although many of the genes involved in the specification of cell identity during this process have been identified, little is known of the genes that coordinate cell movement. Here we show that the zebrafish silberblick (slb) locus1 encodes Wnt11 and that Slb/Wnt11 activity is required for cells to undergo correct convergent extension movements during gastrulation. In the absence of Slb/Wnt11 function, abnormal extension of axial tissue results in cyclopia and other midline defects in the head2. The requirement for Slb/Wnt11 is cell non-autonomous, and our results indicate that the correct extension of axial tissue is at least partly dependent on medio-lateral cell intercalation in paraxial tissue. We also show that the slb phenotype is rescued by a truncated form of Dishevelled that does not signal through the canonical Wnt pathway3, suggesting that, as in flies4, Wnt signalling might mediate morphogenetic events through a divergent signal transduction cascade. Our results provide genetic and experimental evidence that Wnt activity in lateral tissues has a crucial role in driving the convergent extension movements underlying vertebrate gastrulation.

960 citations


Journal ArticleDOI
TL;DR: Understanding to date of how mutations in the APC gene translate into changes at the protein level, which in turn contribute to the role of APC in tumorigenesis are dealt with.
Abstract: Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by the presence of adenomatous polyps in the colon and rectum, with inevitable development of colorectal cancer if left untreated. FAP is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Somatic mutations in the APC gene are an early event in colorectal tumorigenesis, and can be detected in the majority of colorectal tumours. The APC gene encodes a large protein with multiple cellular functions and interactions, including roles in signal transduction in the wnt-signalling pathway, mediation of intercellular adhesion, stabilization of the cytoskeleton and possibly regulation of the cell cycle and apoptosis. The fact that APC is an integral part of so many different pathways makes it an ideal target for mutation in carcinogenesis. This review deals with our understanding to date of how mutations in the APC gene translate into changes at the protein level, which in turn contribute to the role of APC in tumorigenesis.

850 citations


Journal ArticleDOI
20 Dec 1999-Oncogene
TL;DR: A synopsis of current research on Wnt signaling is presented with particular attention paid to molecular mechanism of Wnt signal transduction and how the mis-regulation of WNT signaling leads to cancer.
Abstract: Communication between cells is often mediated by secreted signaling molecules that bind cell surface receptors and modulate the activity of specific intracellular effectors. The Wnt family of secreted glycoproteins is one group of signaling molecules that has been shown to control a variety of developmental processes including cell fate specification, cell proliferation, cell polarity and cell migration. In addition, mis-regulation of Wnt signaling can cause developmental defects and is implicated in the genesis of several human cancers. The importance of Wnt signaling in development and in clinical pathologies is underscored by the large number of primary research papers examining various aspects of Wnt signaling that have been published in the past several years. In this review, we will present a synopsis of current research with particular attention paid to molecular mechanism of Wnt signal transduction and how the mis-regulation of Wnt signaling leads to cancer.

744 citations


References
More filters

Journal ArticleDOI
Andrea H. Brand1, Norbert Perrimon1Institutions (1)
01 Jun 1993-Development
TL;DR: The GAL4 system, a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns, has been designed and used to expand the domain of embryonic expression of the homeobox protein even-skipped.
Abstract: We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.

8,976 citations


"Negative regulation of Armadillo, a..." refers methods in this paper

  • ...Wild-type or mutant cDNAs were introduced into the pUAST vector (Brand and Perrimon, 1993). arm∆N (a.a. 1- 128 deleted) is described by Pai et al. (1996). y arm+ w flies were used for transformation....

    [...]

  • ...To test whether armS10 is dominant lethal, we expressed an armS10 transgene using the inducible GAL-UAS system (Brand and Perrimon, 1993)....

    [...]

  • ...Wild-type or mutant cDNAs were introduced into the pUAST vector (Brand and Perrimon, 1993)....

    [...]

  • ...To test whether armS10 is dominant lethal, we expressed an armS10 transgene using the inducible GAL-UAS system (Brand and Perrimon, 1993). armS10 was cloned downstream of UAS elements regulated by yeast GAL4 and introduced into flies, where it is inactive. armS10 can be activated in particular…...

    [...]


Journal ArticleDOI
30 Oct 1980-Nature
TL;DR: The phenotypes of the mutant embryos indicate that the process of segmentation involves at least three levels of spatial organization: the entire egg as developmental unit, a repeat unit with the length of two segments, and the individual segment.
Abstract: In systematic searches for embryonic lethal mutants of Drosophila melanogaster we have identified 15 loci which when mutated alter the segmental pattern of the larva. These loci probably represent the majority of such genes in Drosophila. The phenotypes of the mutant embryos indicate that the process of segmentation involves at least three levels of spatial organization: the entire egg as developmental unit, a repeat unit with the length of two segments, and the individual segment.

3,936 citations


"Negative regulation of Armadillo, a..." refers background in this paper

  • ...In embryos mutant for either wg signal or for arm, which is required for signal transduction, all cells assume anterior fates and secrete denticles (Nüsslein-Volhard and Wieschaus, 1980)....

    [...]

  • ...We evaluated cell fate choices by examining both cuticle pattern and Wg target gene expression. wg null embryos are shorter than wild-type and secrete only denticles with no naked cuticle (Nüsslein-Volhard and Wieschaus, 1980; Fig....

    [...]

  • ...6 and armXP33 are described by Peifer and Wieschaus (1990), zw3M11-1 is described by Siegfried et al. (1994), and wgIG22 is described by Nüsslein-Volhard and Wieschaus (1980)....

    [...]


Journal ArticleDOI
21 Mar 1997-Science
TL;DR: Results indicate that regulation of β-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or β- catenin.
Abstract: Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by beta-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of beta-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of beta-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or beta-catenin.

3,746 citations


"Negative regulation of Armadillo, a..." refers background in this paper

  • ...Further, mutations in the N terminus of β-catenin very similar to those in ArmS10 are oncogenic in culture (Whitehead et al., 1995) and appear to play a causal role in a variety of different tumors (Kawanishi et al., 1995; Robbins et al., 1996; Morin et al, 1997; Rubinfeld et al., 1997)....

    [...]

  • ..., 1997), and activates transcription via association with Lef-1 or TCF-4 (Rubinfeld et al., 1997; Morin et al., 1997; Korinek et al., 1997)....

    [...]

  • ...…negative regulation (our data; Yost et al., 1996; Munemitsu et al., 1996); point mutants in a putative phosphorylation site within this region have similar effects (Yost et al., 1996; Morin et al, 1997; Rubinfeld et al., 1997), strongly suggesting that phosphorylation is the means of regulation....

    [...]

  • ...Likewise, N-terminally deleted or point mutated β-catenin is stabilized in the soluble pool in mammalian epithelial cells (Munemitsu et al., 1996; Rubinfeld et al., 1997), and activates transcription via association with Lef-1 or TCF-4 (Rubinfeld et al., 1997; Morin et al., 1997; Korinek et al., 1997)....

    [...]

  • ...Regulation of Arm/β-catenin degradation not only plays a key role in Wg/Wnt signaling, but also is a target for activation in both colon cancer and melanoma (Korinek et al., 1997; Morin et al., 1997; Rubinfeld et al., 1997)....

    [...]


Journal ArticleDOI
21 Mar 1997-Science
TL;DR: Constitutive transcription of Tcf target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium.
Abstract: The adenomatous polyposis coli (APC) tumor suppressor protein binds to beta-catenin, a protein recently shown to interact with Tcf and Lef transcription factors. The gene encoding hTcf-4, a Tcf family member that is expressed in colonic epithelium, was cloned and characterized. hTcf-4 transactivates transcription only when associated with beta-catenin. Nuclei of APC-/- colon carcinoma cells were found to contain a stable beta-catenin-hTcf-4 complex that was constitutively active, as measured by transcription of a Tcf reporter gene. Reintroduction of APC removed beta-catenin from hTcf-4 and abrogated the transcriptional transactivation. Constitutive transcription of Tcf target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium.

3,269 citations


"Negative regulation of Armadillo, a..." refers background in this paper

  • ...These mutations dramatically raise the levels of non-junctional β-catenin, driving the formation of complexes with members of the TCF/LEF family and re-programming gene expression (Korinek et al, 1997; Rubinfeld et al., 1997)....

    [...]

  • ..., 1997), and activates transcription via association with Lef-1 or TCF-4 (Rubinfeld et al., 1997; Morin et al., 1997; Korinek et al., 1997)....

    [...]

  • ...Likewise, N-terminally deleted or point mutated β-catenin is stabilized in the soluble pool in mammalian epithelial cells (Munemitsu et al., 1996; Rubinfeld et al., 1997), and activates transcription via association with Lef-1 or TCF-4 (Rubinfeld et al., 1997; Morin et al., 1997; Korinek et al., 1997)....

    [...]

  • ...Regulation of Arm/β-catenin degradation not only plays a key role in Wg/Wnt signaling, but also is a target for activation in both colon cancer and melanoma (Korinek et al., 1997; Morin et al., 1997; Rubinfeld et al., 1997)....

    [...]

  • ...…N-terminally deleted or point mutated β-catenin is stabilized in the soluble pool in mammalian epithelial cells (Munemitsu et al., 1996; Rubinfeld et al., 1997), and activates transcription via association with Lef-1 or TCF-4 (Rubinfeld et al., 1997; Morin et al., 1997; Korinek et al., 1997)....

    [...]


Journal ArticleDOI
Jürgen Behrens1, von Kries Jp1, Michael Kühl2, Bruhn L3  +5 moreInstitutions (4)
15 Aug 1996-Nature
TL;DR: β-catenin regulates gene expression by direct interaction with transcription factors such as LEF-1, providing a molecular mechanism for the transmission of signals from cell-adhesion components or wnt protein to the nucleus.
Abstract: The cytoplasmic proteins beta-catenin of vertebrates and armadillo of Drosophila have two functions: they link the cadherin cell-adhesion molecules to the cytoskeleton, and they participate in the wnt/wingless signal pathway. Here we show, in a yeast two-hybrid screen, that the architectural transcription factor LEF-1 (for lymphoid enhancer-binding factor) interacts with beta-catenin. In mammalian cells, coexpressed LEF-1 and beta-catenin form a complex that is localized to the nucleus and can be detected by immunoprecipitation. Moreover, LEF-1 and beta-catenin form a ternary complex with DNA that splays an altered DNA bend. Microinjection of LEF-1 into XenoPus embryos induces axis duplication, which is augmented by interaction with beta-catenin. Thus beta-catenin regulates gene expression by direct interaction with transcription factors such as LEF-1, providing a molecular mechanism for the transmission of signals, from cell-adhesion components or wnt protein to the nucleus.

2,704 citations


"Negative regulation of Armadillo, a..." refers background in this paper

  • ...…can form a complex with DNA-binding proteins of the TCF/LEF family, and these proteins could, when ectopically expressed in Xenopus, alter dorsal-ventral patterning in a way which suggested that they play a role in Wnt signaling (Behrens et al., 1996; Molenaar et al., 1996; Huber et al., 1996)....

    [...]


Network Information
Performance
Metrics
No. of citations received by the Paper in previous years
YearCitations
20213
20206
20192
201813
20175
20166