|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online May 23, 2006
doi: 10.1242/10.1242/dev.02398
1 Department of Biological Sciences, Carnegie Mellon University, 4400 5th
Avenue, Pittsburgh, PA 15213, USA.
2 Department of Biology, University of North Carolina at Chapel Hill, CB# 3280
Coker Hall, Chapel Hill, NC 27599, USA.
3 Curriculum in Genetics and Molecular Biology, University of North Carolina at
Chapel Hill, CB# 3280 Coker Hall, Chapel Hill, NC 27599, USA.
4 Department of Biology, Duke University, Durham, NC 27710, USA.
5 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel
Hill, CB# 3280 Coker Hall, Chapel Hill, NC 27599, USA.
Authors for correspondence (e-mail:
brookem{at}andrew.cmu.edu;
peifer{at}unc.edu)
Accepted 7 April 2006
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Mutation cluster region, Familial adenomatous polyposis, Adherens junctions, ß-Catenin
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), while the Wnt regulator APC is the tumor suppressor mutated
in the hereditary colon cancer syndrome familial adenomatous polyposis (FAP)
(reviewed by Gaspar and Fodde,
2004). APC mutations also underlie >70% of sporadic
colon cancers.
APC encodes a multidomain protein with several functions
(Fig. 1A). It regulates Wnt
signaling as part of the `destruction-complex', which maintains low levels of
cytoplasmic ß-catenin, the key Wnt effector, in the absence of Wnt
signals (reviewed by Polakis,
2000). Within this complex, APC binds both ß-catenin, via its
15- and 20-amino acid repeats, and the scaffolding protein Axin, via its SAMP
repeats (Fig. 1A). Axin and APC
present ß-catenin to Casein kinase I and GSK3ß (fly Zw3), which
sequentially phosphorylate ß-catenin, targeting it for ubiquitination and
destruction. Interestingly, most colon tumors retain an APC protein truncated
in the `mutation-cluster region' (MCR, Fig.
1A), lacking the SAMP repeats and all sequences located between
that region and the C-terminal end
(Polakis, 2000). Wnt ligands
like Drosophila Wingless (Wg) inactivate the destruction-complex and
stabilize ß-catenin by engaging a Frizzled/LRP5/LRP6 receptor complex.
The mechanism of destruction-complex inactivation is not well understood, but
involves Dishevelled and interactions between LRP5/6 and Axin. Wnt signals may
alter Axin localization or stability
(Cliffe et al., 2003;
Tolwinski et al., 2003).
Both mammals and Drosophila have two APC proteins with shared and
divergent structures (Fig. 1).
One key question about APC function concerns the relative roles of family
members. Mammalian APC is broadly expressed and homozygous mutants die during
gastrulation (Moser et al.,
1995). Mammalian APC2 is strongly expressed in the CNS
(Nakagawa et al., 1998;
van Es et al., 1999;
Yamanaka et al., 2002), but
its mutant phenotype has not been reported. Drosophila APC1 is
strongly expressed in the CNS and germline, and homozygous mutants are viable
and fertile with defects confined to eye development
(Ahmed et al., 1998;
Hayashi et al., 1997).
Drosophila APC2 is broadly expressed. Zygotic mutants are viable and
normal, but maternal/zygotic (M/Z) mutants die with defects in Wg signaling
during embryogenesis (McCartney et al.,
1999). Fly APC1 and APC2 are partially redundant in post-embryonic
Wg signaling and Wg-independent brain development
(Ahmed et al., 2002;
Akong et al., 2002a;
Akong et al., 2002b). This
occurs despite the fact that their domain structures and subcellular
localizations are distinct. APC1 carries the basic domain, and localizes to
centrosomes and microtubules, whereas APC2 lacks that domain and localizes to
the cortex (Akong et al.,
2002b).
In addition to regulating Wnt signaling, APC family proteins have proposed
functions in cytoskeletal regulation (reviewed by
Nathke, 2004). These are
reflected in its binding partners (Fig.
1A). The N-terminal third of APC encodes a series of
Armadillo (Arm) repeats, binding sites for cytoskeletal regulators, including
the Rac-GEF ASEF, kinesin-associated KAP3, and IQGAP. The C-terminal third
encodes a basic region that binds microtubules and the formin Diaphanous
(Wen et al., 2004), and a
binding site for the microtubule plus-end-binding protein EB1.
Many different cytoskeletal functions have been proposed for APCs.
Loss-of-function studies using putative hypomorphic alleles suggest that APC2
helps mediate interactions between mitotic spindles and cortical actin in
early embryos (McCartney et al.,
2001), and works with APC1 to regulate mitotic spindle orientation
in the male germline (Yamashita et al.,
2003). Certain APC2 alleles also affect cadherin-based
adhesion (Hamada and Bienz,
2002; Townsley and Bienz,
2000). RNAi of APC2 alters the symmetric divisions of
ectodermal epithelial cells (Lu et al.,
2001). Studies in cultured cells using either truncations or
dominant-negative approaches suggest roles for mammalian APC in kinetochore
function (Fodde et al., 2001;
Kaplan et al., 2001) and
microtubule organization in polarized cells
(Etienne-Manneville and Hall,
2003; Kawasaki et al.,
2003; Shi et al.,
2004; Wen et al.,
2004; Zhou et al.,
2004). Finally, in vitro studies suggest APC functions in spindle
assembly (Dikovskaya et al.,
2004). However, in many cases these effects were subtle. Because
these studies did not use null alleles, and some used `dominant-negative'
approaches, two distinct possibilities remain: (1) APC family proteins may
play essential roles in some of these cytoskeletal processes, which were not
fully disrupted using the partial loss-of-function approaches employed; or (2)
APC family proteins may be non-essential for some of these processes, but the
truncated APC proteins expressed by the mutant alleles or the
`dominant-negative' constructs may disrupt them because of their ability to
bind and inactivate other essential players. Consistent with the latter
possibility, siRNA inactivation of mammalian APC led to less severe spindle
defects than those observed in a truncation mutant
(Green et al., 2005).
|
) included a
protein-null allele, the only tissue defective in single mutants is the eye,
where loss of APC1 triggers apoptosis, precluding assessment of other cell
biological roles.
A second striking but unanswered question is why, although both copies of
APC are invariably mutated in colon tumors, one allele encodes a
truncated APC protein ending in the MCR
(Fig. 1A). This contrasts with
most tumor suppressors. The truncated proteins cannot correctly regulate
ß-catenin, although whether they are null for this function is unclear.
Data from tumors and engineered mouse mutations
(Smits et al., 1999) suggest
that truncated proteins are selected for the deletion of all Axin-binding SAMP
repeats, reducing their ability to regulate Wnt signaling. While this explains
the function lost by truncation, it does not explain why truncations are
inevitable. Bi-allelic null mutations would eliminate function, but are not
found in human tumors. There is substantial controversy over whether the
truncated proteins have dominant effects or simply reduce APC function. We
used genetic and cell biological tools in Drosophila to address these
crucial questions.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and crossed
to females carrying TM3 Sb. Individual F1 males carrying the
mutagenized chomosome (*) over TM3 Sb were crossed to
virgin APC2
S females at
27°C (its non-permissive temperature). Embryos maternally and zygotically
APC2 mutant die as embryos, whereas paternally rescued embryos
survive to adulthood. In crosses where the mutagenized chromosome carried a
new allele of APC2, half of the progeny should die as embryos. After
three days, each vial was scored for the presence of unhatched embryos whose
cuticles had turned brown; these arise when eggs are fertilized but embryos
die after having produced cuticle. Approximately 9400 fertile crosses were
scored in six rounds of mutagenesis. For lines that exhibited significant
embryonic lethality, males were recovered from the crosses and individually
crossed to virgin females carrying TM6 Tb to recover the mutagenized
chromosome. 110 lines were generated and re-tested for the failure to
complement APC2S. Nineteen
lines failed to complement
APC2S a second time and the
mutant embryos exhibited cuticle phenotypes consistent with activation of Wg
signaling. Of these mutant chromosomes, we detected molecular lesions in seven
(APC2b5, APC2c9, APC2d40,
APC2e90, APC2f90, APC2g10 and
APC2g41) upon sequencing the APC2 gene from
PCR-amplified genomic DNA from APC2-/Df(3R) crb 87-4 adults.
Hatch rates and cuticle analysis
Embryos were the progeny of APC2allele/Df(3R)crb 87-4 females
x APC2allele/+ males. All alleles were scored at 27°C and
18°C. Double-mutant embryos maternally FRT APC2allele
APC1Q8 were generated using the FRT/FLP/DFS technique
(Chou and Perrimon, 1996).
Cuticle preparations and hatch rate analysis were as described by Wieschaus
and Nusslein-Volhard (Wieschaus and
Nusslein-Volhard, 1998).
Immunolocalization, immunoblots and imaging
Embryos and ovaries were from adults maintained at 27°C. Tissues were
fixed as described previously (McCartney
et al., 1999). Antibodies/probes used were as follows: for actin,
Alexa-488-phalloidin (1:500, Molecular Probes); rat anti-APC2-CT (1:1000)
(McCartney et al., 1999);
mouse anti-tubulin-E7 (1:1000); anti-Arm-N27A1 (1:500); anti-DE-cadherin-DCAD2
(1:50); anti-En (1:50, all Developmental Studies Hybridoma Bank); for DNA,
propidium iodide (25 µg/ml, Molecular Probes) or DAPI (10 µg/ml,
Sigma-Aldrich). DAB reactions were carried out as described by McEwen et al.
(McEwen et al., 2000).
Anti-APC2-NT antisera were raised in guinea pigs by Pocono Rabbit Farms &
Laboratory (Canadenisis, PA) against GST-APC2 (amino acids 1-126).
Immunoblotting used anti-APC2 at 1:1000, detected using the SuperSignal West
Pico Chemiluminescent Substrate (Pierce). For nuclear loss, DAPI-stained
nuclei near the cortex but below the cortical monolayer of nuclei were scored
as lost - one surface of each embryo was analyzed. The percentage of nuclei
lost was determined as the number of lost nuclei/total number of nuclei
(estimated from nuclear number in 1 µm2 of the cortex).
Imaging was performed using various microscope systems. The images in Figs 4, 5 were produced using a Zeiss LSM510 with gain, offset, and pinhole size kept constant for each wavelength; those in Fig. 6 and Fig. 9A-H used a Zeiss LSM510 or LSM410, respectively, with internal wild-type controls expressing histone-GFP co-stained with mutants to control for staining variations. The images in Fig. 7A-C,H-J were produced using a Spinning-disc confocal microscope (Solamere Technology Group) with a Yokogawa scanhead and a Hamamatsu OrcaAG CCD camera on a Zeiss Axiovert200M; those in Fig. 7D,E and Fig. 8 used a Zeiss Axioskop2 Plus and a Canon Powershot G5 camera. Analysis of Fig. 6E'',F'' and Fig. 7F,G,K used Image-J. Figures were prepared using Adobe PhotoShop.
APC2 rescuing transgene
Genomic DNA (688 bp) from the gene upstream of APC2 (CG13608) to
the AUG (the `endogenous promoter') of APC2 was PCR amplified and
inserted into the EcoRI site of pCaSpeR-2 (modified to include the
ADH-polyadenylation signal of pRmHa-3). The APC2 coding region (cDNA
LD18122) was inserted 3' to the endogenous promoter. P-element-mediated
transformation generated several lines. Two independent second-chromosome
lines rescued the maternal-effect lethality of
APC2S at 27°C (data not
shown).
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
S
(McCartney et al., 1999). This
screen would allow us to identify null alleles, as deficiencies removing
APC2 fail to complement
APC2S.
We obtained seven mutations affecting the coding region of APC2
(Fig. 1C). Three are missense
mutations in the Arm repeats (APC2e90 repeat 3,
APC2c9 repeat 6, and APC2b5 repeat 7;
altered amino acids are shown in Fig.
1C). Four alleles result from premature stop codons that should
truncate the protein. Two truncate APC2 within Arm repeats 8 or 9:
APC2g10 and APC2f90. Two others
truncate APC2 within the MCR: APC2d40 [amino acid 677; its
phenotype has been reported previously
(Ahmed et al., 2002;
Akong et al., 2002a;
McCartney et al., 2001)] and
APC2g41 (amino acid 728). Together with
APC2S
(McCartney et al., 1999),
which deletes a single serine in Arm repeat 5, and
APC2N175K, identified by Hamada and Bienz
(Hamada and Bienz, 2002), a
missense mutation in Arm repeat 3, there are nine APC2 alleles.
Several mimic those in human tumors or those engineered in mice.
APC2f90 resembles mouse APC716 and
APCMIN that truncate just C terminal to the Arm repeats
(Oshima et al., 1995a;
Su et al., 1995). The
APC2d40 and APC2g41 truncations are
similar to those in the human APC MCR.
|
). To determine whether APC2f90 and
APC2g10 are protein null, we used antibodies against an
N-terminal APC2 epitope (amino acids 1-126,
Fig. 1C) that would be present
in the predicted truncated proteins. This antibody recognized background bands
at 59 and 44 kDa, and recognized strongly reduced levels of a 41 kDa truncated
protein in APC2f90
(Fig. 3C), close to its
predicted molecular weight (Table
1). No APC2g10 protein at or near the expected
size was detected (Fig. 3C),
suggesting that APC2g10 is protein null. Consistent with
this, it is among our strongest alleles (see below), and its cuticle phenotype
over a deficiency resembles its homozygous phenotype (data not shown), the
genetic test for a null allele.
|
Both the Arm repeats and the C terminus are required for cortical association
APC2 localizes to the cortex, co-localizing with cortical actin and
overlapping adherens junctions (Fig.
4A) (McCartney et al.,
1999; Yu and Bienz,
1999; Yu et al.,
1999). We used our mutants to determine whether particular domains
are essential or dispensable for cortical localization (we did not examine
APC2f90 and APC2g10, as anti-APC2-NT
does not work in tissue). APC2S
protein fails to localize to the cortex
(McCartney et al., 1999),
implicating Arm repeat 5 in correct localization. APC2c9,
affecting Arm repeat 6, and APC2b5, affecting repeat 7,
encode exclusively cytoplasmic proteins (data not shown), supporting a role
for Arm repeats 5-7 in localization. The two proteins truncated in the MCR,
APC2d40 and APC2g41, are also
exclusively cytoplasmic (Fig.
4C; data not shown), supporting a role for the C terminus of APC2
(including the Axin-binding sites) in cortical localization.
APC2e90 and APC2N175K proteins weakly
associate with the cortex (Fig.
4B'; data not shown).
|
S mutants exhibit reduced
Arm at adherens junctions, resulting in reduced cadherin-based adhesion,
although this effect was not as dramatic as eliminating essential junctional
proteins (Hamada and Bienz,
2002; Townsley and Bienz,
2000). One hypothesis is that eliminating APC2 and
APC1 function might disrupt adhesion completely.
We first tested this in ovaries. Each egg chamber has 15 nurse cells and an
oocyte surrounded by the somatic follicular epithelium
(Fig. 5A,C). Arm normally
accumulates strongly in follicle cell adherens junctions, and weakly at nurse
cell junctions (Peifer et al.,
1993). We examined females null for APC2 and reduced for
APC1 function (APC2g10
APC1Q8/APC2g10), thus reducing function equally in
both germ cells and somatic follicle cells, and also egg chambers in which the
germline was homozygous APC2g10 APC1Q8
(Fig. 5B,D,F; data not shown).
In some mutant egg chambers, cytoplasmic Arm levels were elevated
(Fig. 5B',D'),
consistent with a loss of destruction-complex function. However, cortical Arm
localization was largely unaltered in mutant germ cells and follicle cells,
regardless of whether (Fig.
5B',D') or not
(Fig. 5F') they had
elevated Arm levels.
|
). In APC2S or
APC2N175K mutants, oocytes are occasionally mispositioned
(Hamada and Bienz, 2002;
Townsley and Bienz, 2000). To
determine the effect of loss of APC function on oocyte position, we compared
wild type, APC2g10, APC2g10
APC1Q8/APC2g10, and APC2g10
APC1Q8 mutant germlines. We distinguished the oocyte from
nurse cells by the presence of four ring canals
(Fig. 5B, arrow) and its
elevated accumulation of cortical actin. There was no difference in the
frequency of mispositioned oocytes between wild type and mutants
(Fig. 5E). Thus a complete loss
of APC function does not substantially reduce cadherin-based adhesion in
ovaries.
Embryonic adherens junctions are established during cellularization, and
total loss of cadherin-based adhesion results in catastrophic defects in
epithelial architecture (Cox et al.,
1996; Tepass et al.,
1996). We did not observe defects in epithelial structure in M/Z
APC2 APC1 double null embryos
(Fig. 6B,D,F). Furthermore, we
did not observe disruption of cortical localization of DE-cadherin
(Fig. 6A,B) or 
-catenin
(Fig. 6C-F) in embryonic
epithelia. In fact, if anything, cortical localization was slightly elevated.
Arm levels were highly elevated (Fig.
6B,D), but cortical Arm was still present. Finally, the cuticles
of these embryos (see below) did not display the total disruption seen when
adhesion is strongly compromised. The discrepancy with the earlier results of
the Bienz Laboratory suggests that the proteins they tested may have
dominant-negative effects on cadherin-catenin function; consistent with this,
APC2S has dominant-negative
effects on cortical nuclear retention (see below).
Assessing roles of APC proteins in nuclear retention, spindle morphology and orientation
After fertilization, Drosophila embryos undergo a series of
nuclear divisions without cytokinesis. As microtubules form spindles, actin
lines transient furrows separating adjacent nuclei, preventing spindle
collisions (Fig. 7A). Thus,
defects in actin or microtubule function can result in abnormal nuclear
divisions with resulting abnormal nuclei transported into the embryo interior.
In APC2S mutants, a subset of
peripheral nuclei are lost without significant actin or microtubule defects
(McCartney et al., 2001),
suggesting that APC2 helps tether actin to microtubules, thereby tethering
nuclei to the cortex.
|
|
S
and APC2d40 (McCartney
et al., 2001) was enhanced. We quantified nuclear loss in
APC2 mutant embryos or in embryos doubly null for APC2 and
APC1 (Fig. 7F). We
defined an abnormal embryo as one in which 
2% of the cortical nuclei have
moved into the embryo interior (Sullivan
et al., 1993). In our two wild-type strains (y w and
Oregon-R), 0-3% of embryos are abnormal, consistent with previous reports
(2-3%) (Sullivan et al.,
1993).
We first examined the effects of a complete lack of APC2.
Thirty-five percent of APC2g10 maternally mutant embryos
are abnormal (Fig. 7E,F). This
is partially rescued by P[APC2+], an APC2
transgene driven by the endogenous promotor (14%;
Fig. 7F); this incomplete
rescue may be due to reduced levels of APC2 expression from the transgene.
Seven percent of the progeny of mothers heterozygous for
APC2g10 are abnormal - this may reflect slight
haploinsufficiency or may be within the wild-type range. To determine whether
APC1 also has syncytial functions, we examined nuclear loss in
APC2g10 APC1Q8 maternally double-mutant embryos
(Fig. 7B,C,F). Thirty-nine
percent of the embryos were abnormal, similar to APC2g10
alone (35%), suggesting that if APC1 functions in this process, its
contribution is relatively minor. In
APC2S, nuclear loss appears to
be largely due to disrupted tethering
(McCartney et al., 2001). In
APC2g10 and in double null mutants, this may be compounded
by spindle collisions resulting from compromised actin furrows
(Fig. 7B,B', arrow).
However, even in the most severe cases, many nuclei remain at the cortex.
Our mutant alleles also allowed us to assess which domains of APC2 are
important for function in nuclear retention, and to examine whether truncated
proteins have dominant effects on this process. We previously documented
nuclear retention defects in APC2d40
(McCartney et al., 2001),
which is truncated in the MCR (Fig.
1). Eighteen percent of APC2d40 maternally
mutant embryos are abnormal (Fig.
7F). Interestingly, progeny of APC2d40
heterozygous mothers had a similar phenotype (19% abnormal), suggesting that
APC2d40 has dominant-negative effects on this process.
We also assessed nuclear loss for three missense mutations affecting the
Arm repeats (APC2N175K, APC2c9 and
APC2S;
Fig. 1C,
Fig. 7F).
APC2N175K and APC2c9 maternal mutants
have relatively weak phenotypes (12% and 8% abnormal embryos). By contrast,
58% of APC2S embryos are
abnormal. This is reduced to 15% by P[APC2+], which
rescues the null allele to the same degree. This indicates that the dramatic
effect of APC2S is due to its
effect on APC2. In progeny of mothers heterozygous for
APC2S, the frequency of
abnormal embryos (21%) was substantially higher than in wild type (2-3%), and
was much higher than that caused by heterozygosity for the null allele (7%).
This suggests that APC2S protein, like APC2d40, is
dominant negative in this process.
|
). In
syncytial Drosophila embryos, thousands of nuclei divide
synchronously, providing an excellent system in which to examine whether fly
APCs are crucial for spindle structure. In
APC2S mutants, nuclei and
spindles are lost from the cortex without significant defects in spindle
morphology (McCartney et al.,
2001). To determine whether complete loss of APC2 and
APC1 affects syncytial spindles, we compared spindle morphology in
embryos maternally APC2g10 APC1Q8 double mutant
with nuclear- and cell-cycle stage-matched wild types. APC2 APC1 null
embryos did not have significant defects in overall spindle morphology
(Fig. 7B',C').
Astral microtubules are not easily observed in syncytial embryos
(Foe et al., 1993), and thus
we did not examine them. The one change in spindles we noted was a slight but
significant lengthening of the pole-to-pole distance
(Fig. 7G).
After cellularization, ectodermal cells divide in synchronous regions
called mitotic domains (Foe et al.,
1993). We next compared spindle orientation and division plane in
wild-type and APC2g10 APC1Q8 M/Z mutant
ectoderm. Wild-type spindles are oriented perpendicular to the apical-basal
axis (92%, Fig. 7H,K) and
divisions are symmetric, resulting in two equal daughter cells (100%,
Fig. 7H,K). A previous RNAi
study (Lu et al., 2001)
suggested that disrupting APC2 function results in mis-oriented spindles and
asymmetric cytokinesis in the ectoderm. However, spindles in
APC2g10 APC1Q8 M/Z mutant embryos are oriented
normally (90-92%, Fig. 7J,K),
and all divisions were symmetric (100%,
Fig. 7J,K). We also examined
whether truncated APC2 proteins have dominant effects, examining divisions in
APC2d40/+ embryos derived from APC2d40
homozygous mothers. We observed no dominant-negative effects
(Fig. 7I,K). One possible
explanation of the discrepancy with the results of Lu et al.
(Lu et al., 2001) is
off-target RNAi effects of their 1 kb dsRNA. Thus, APC family proteins do not
play a key role in spindle structure, orientation or division plane selection
in the Drosophila ectodermal epithelium.
Relating APC2 structure to function in Wnt signaling
APC proteins play an essential role in regulating Wnt signaling, but key
questions remain about the relationship between structure and function. We
used our APC2 mutations to assess the requirement for different
domains in Wg regulation, and to determine the level of function retained by
truncated proteins and their potential for dominant-negative effects on Wg
signaling
The embryonic cuticle provides a sensitive readout of cell fate choices. We
examined the cuticle phenotype of M/Z mutants, placing each allele in trans to
a deletion of APC2 to reduce concerns about other background
mutations. We initially assessed phenotypes at 27°C, in case any mutations
were temperature sensitive. Global activation of Wg signaling has several
consequences for embryogenesis. Three phenotypes vary roughly in parallel. (1)
Epidermal cell fates - increased Wg signaling leads to fewer
denticle-producing cells and more smooth cuticle
(Fig. 8D, black arrow). (2)
Cuticle size - elevated Wg signaling results in fewer epidermal cells, due to
apoptosis (Pazdera et al.,
1998). (3) Head morphology - elevated Wg signaling disrupts head
involution (Fig. 8D, open
arrow). We scored >180 embryos per genotype, assigned each to a phenotypic
category from weak (0) to strong (6; representative cuticles are in
Fig. 8), and calculated a
phenotypic average (pa) for each allele. The nine APC2 alleles form a
phenotypic series with phenotypic averages ranging from 2.0 for
APC2e90 to 4.5 for APC2g10
(Table 1). This divided the
alleles into three categories based on Wg activation: (1) weak -
APC2e90, APC2b5 and
APC2N175K; (2) moderate - APC2c9,
APC2S and
APC2d40; and (3) strong - APC2g41,
APC2f90 and APC2g10 [our
APC2N175K stock has a somewhat weaker phenotype than had
been previously reported (Hamada and
Bienz, 2002)].
We also assessed effects on Wg signaling directly, examining Arm stability.
In wild type, Arm accumulates at cell-cell junctions in all cells, whereas in
cells receiving Wg Arm also accumulates in the cytoplasm and nucleus, due to
inactivation of the destruction-complex
(Fig. 9A).
APC2S mutants have elevated Arm
levels (McCartney et al.,
1999), but not as elevated as those seen when the
destruction-complex is completely inactivated by M/Z loss of Zw3 kinase
(Peifer et al., 1994;
Siegfried et al., 1994). The
less severe phenotype of APC2S
is due, in part, to slight APC1 activity
(Ahmed et al., 2002;
Akong et al., 2002a), but might
also suggest that APC2S does
not fully inactivate APC2.
The cuticle phenotypes of our APC2 alleles correlate well with Arm
levels. The weakest allele, APC2e90, has only a slight
elevation in Arm levels; stripes are still readily apparent
(Fig. 9B). Other weak to
moderate alleles with missense mutations in the Arm repeats
(APC2c9 and APC2b5) have a slightly
greater elevation of interstripe Arm levels, but stripes remain detectable
(Fig. 9C,D) - they resemble
APC2S
(McCartney et al., 1999). The
alleles truncating APC2 in the MCR, APC2d40
(Fig. 9E)
(Akong et al., 2002a) and
APC2g41 (Fig.
9F), are stronger still, showing uniformly high levels equivalent
to those in wild-type Arm stripes. Finally, APC2f90 and
the null allele APC2g10 accumulate Arm levels in all cells
higher than those in wild-type stripes
(Fig. 9G,H). The highest Arm
levels, however, are only seen when APC1 is also removed, in
APC2g10 APC1Q8 double null mutants
(Fig. 9I) - these resemble the
extremely high levels previously seen in APC2d40
APC1Q8 double mutants
(Ahmed et al., 2002;
Akong et al., 2002a) and in
zw3 M/Z mutants (Peifer et al.,
1994; Siegfried et al.,
1994) (Fig. 9J). We
also examined the expression of a Wg target gene, engrailed
(en), in our strongest alleles, APC2g41,
APC2f90 and APC2g10. en is activated in
additional cells posterior to its normal domain, but it is not activated in
all cells (see Fig. S1 in the supplementary material), results that are
identical to what was previously observed in
APC2S
(McCartney et al., 1999) or
M/Z zw3 mutants (Siegfried et
al., 1992).
|
|
S is temperature
sensitive, exhibiting M/Z embryonic lethality at 25°C and viability at
18°C (McCartney et al.,
1999). To determine whether other APC2 alleles are
temperature sensitive, we assessed their cuticle phenotypes and hatch rates at
18°C (Table 1). Strikingly,
all mutants with missense mutations in the Arm repeats, except
APC2N175K, are temperature sensitive; many M/Z mutants
hatch as larvae. By contrast, the truncation alleles APC2d40,
APC2g41 and APC2f90, and the null allele
APC2g10 are not temperature sensitive.
We also assessed whether mutants had dominant-negative effects on Wg
signaling. None affect adult patterning in zygotic heterozygotes or
homozygotes, ruling out strong dominant-negative effects. As a more sensitive
test, we assessed paternal rescue of M/Z embryonic lethality, which requires
that paternal wild-type APC2 can counter the effects of M/Z mutant protein. If
paternal rescue is fully effective, 50% of the progeny of mutant females
crossed to heterozygous males should hatch. For eight alleles, 
50%
(46-52%, Table 1) of the
offspring hatch as larvae. The exception is APC2c9, where
only 38% hatch. Thus, most alleles, including those producing proteins
truncated in the MCR, do not have apparent dominant-negative effects on Wg
signaling; however, APC2c9 may have some dominant effect
on viability, such that paternal rescue is incomplete.
APC1 acts redundantly with APC2 in Wg regulation in many tissues
(Ahmed et al., 2002;
Akong et al., 2002a;
Akong et al., 2002b). In
embryos, low levels of APC1 in the epidermis provide a small amount of
residual function when APC2 is reduced. Interestingly, the cuticle phenotypes
of the strongest APC2 single mutants
(Fig. 8) are roughly as severe
as those of APC2d40 APC1Q8 double mutants
(Ahmed et al., 2002;
Akong et al., 2002a), although
their effects on Arm levels (Fig.
9) suggest residual APC1 function in APC2 null single
mutants. To further explore this, we examined the cuticle phenotypes of
embryos M/Z double mutant for a null allele of APC1
(APC1Q8) and several APC2 alleles
(Table 1). All show
approximately the same severity of cuticle phenotype [phenotypic average
(pa)=3.7-4.0; Fig. 8H,I],
suggesting that, in the absence of APC1, all are so disabled that Wg
regulation drops below the threshold of function measurable in our cuticle
assay. Alternatively, the partial activity of some mutant proteins may depend
on APC1 function in some way. APC2g10 APC1Q8
double null mutants (pa=3.8, n=238;
Fig. 8I) are quite similar to
axin (axn) null embryos (pa=4.1, n=239;
Fig. 8J), which fully
inactivate the destruction complex. In fact, this analysis slightly
underestimates the double null phenotype, because some paternally rescued
embryos die (data not shown). APC2g10 APC1Q8
M/Z mutants selected using a GFP marker have a more severe phenotype (pa=4.7,
n=96).
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Assessing roles for APC proteins in cell adhesion and spindle function
Experiments in vitro, in cultured cells and in Drosophila
suggested novel roles for APCs in cadherin-based adhesion
(Hamada and Bienz, 2002;
Townsley and Bienz, 2000),
spindle structure and chromosome segregation (e.g.
Green and Kaplan, 2003).
Although some of these effects were subtle, APC family function was not
completely eliminated, suggesting that APCs may play essential roles in one or
more of these processes. Alternatively, because these phenotypes were assessed
in cells expressing truncated or otherwise mutant proteins, or expressing
transfected APC fragments, it is possible that these effects result from
dominant interference with binding partners of APC that work in a process in
which APC proteins themselves are not essential.
To distinguish between these possibilities, null mutations removing the
function of both APCs must be characterized. In mammals, all work has been
done in single mutants and most was done with cells or animals expressing one
truncated APC allele. Recently, Cre-lox technology was used to
generate mouse APC alleles that may be null; these delete exon 14,
and are predicted to truncate APC before the Arm repeats
(Colnot et al., 2004;
Shibata et al., 1997).
Although the phenotype of homozygous animals has not been reported, Cre
induction was used to create homozygous mutant clones of colon cells. This
triggers polyp formation (Shibata et al.,
1997), with mutant cells assuming stem cell properties consistent
with Wnt activation (Andreu et al.,
2005; Sansom et al.,
2004). Other phenotypes were not assessed, however, and tests to
confirm that this allele is protein null were not reported, so splicing
variations might produce residual mutant protein.
We examined ovaries and embryos null for APC2, or double null for
both APC2 and APC1, for essential roles in cadherin-based
adhesion. We did not observe phenotypes consistent with substantial disruption
of cadherin-catenin function, which disrupts both oogenesis and embryonic
epithelial integrity (Cox et al.,
1996; Tepass et al.,
1996). In ovaries, loss of APC2 and APC1 had no
apparent effect on adhesion, and in embryos we did not observe significant
alterations in DE-cadherin or -catenin localization at adherens
junctions. Thus APC family proteins do not play an essential role in cell
adhesion. However, we cannot rule out subtle modulatory effects.
We also tested proposed roles for APC proteins in spindle assembly and
orientation. Embryos null for both APC proteins had no defects in spindle
structure in syncytial embryos, apart from those in regions of spindle
detachment or defective metaphase furrows, and no defects in spindle
orientation or cell division symmetry in the ectoderm during gastrulation.
Thus APC family proteins are not essential for spindle function in these
tissues. We did see a subtle but significant lengthening of syncytial spindles
during cycle 13. We did not assess subtle defects in chromosome segregation,
which might lead to a slow accumulation of aneuploid cells in tumors - this
will require other assays. How can we reconcile the earlier data suggesting
that APC family proteins have roles in adhesion and cytoskeletal regulation
when our full loss-of-function experiments indicate that they do not? One
possibility is that truncated fragments of APC may have dominant effects on
processes in which APC does not play an essential role - our data on the
phenotype of APC2S in spindle
tethering, which is discussed in more detail below, provide an example of
this.
Are truncated APC proteins dominant negative?
Unlike most other tumor suppressors, APC homozygous null colon
tumors are either rare or non-existent. Instead, one allele encodes a protein
truncated in the MCR, suggesting strong selection for this event during tumor
development. Several models propose that the truncated APC proteins found in
tumors are dominant negative. One suggests that this affects Wnt signaling,
with truncated APC proteins promoting stem cell proliferation
(Kim et al., 2004). Most
models suggest that truncated proteins affect cytoskeletal functions.
Different studies come to different conclusions, however. For example, some
suggest that truncated APC interferes with microtubule-kinetochore
attachments, leading to genomic instability
(Green and Kaplan, 2003;
Green et al., 2005;
Tighe et al., 2001), but
others suggest these effects are subtle
(Sieber et al., 2002a). A
dominant-negative role of truncated APC is not essential for disease, as some
FAP patients inherit germline-null APC mutations
(Laken et al., 1999;
Sieber et al., 2002b). Their
adenomas carry truncating mutations in the other allele; in this case, there
was no wild-type APC to be affected by a dominant-negative truncation.
Furthermore, the putative dominant-negative effect is not sufficient for
oncogenesis - mice engineered to express truncated APC in a wild-type
background do not develop polyps or tumors
(Oshima et al., 1995b).
Our genetic data provide new insight into this question. We saw little evidence for dominant-negative effects on Wg signaling. Heterozygotes are viable and adults are wild type in phenotype, and wild-type paternal APC2 effectively rescues eight of the nine mutants, suggesting that mutant proteins cannot be strongly dominant negative. The exception is APC2c9, where there appears to be some interference with paternal rescue. Likewise, our data suggest that truncated APC2 does not substantially affect spindle structure (we did not address whether APC1 truncations behave dominantly).
We did, however, find compelling evidence for dominant-negative effects on
nuclear retention in syncytial embryos.
APC2S and
APC2d40 heterozygotes exhibit elevated levels of nuclear
loss, and the frequency of abnormal embryos is higher in
APC2S homozygotes than in
APC2 null mutants. Thus, the cytoskeletal functions of APCs may be
more sensitive to dominant-negative effects of truncated proteins, and this
may affect chromosome segregation and contribute to tumor progression.
Our data also illuminate the mechanisms of dominant-negative activity. Loss
of APC1 did not enhance the nuclear-loss phenotype of APC2
null embryos, suggesting that APC1 does not play a significant role in this
process. This suggests that the dominant-negative effect is not on maternally
contributed APC1 (Hayashi et al.,
1997). As nuclear retention is not completely disrupted in double
null mutants, alternative mechanisms of nuclear retention partially compensate
for the lack of APC1 and APC2. Because
APC2S has a nuclear retention
defect more severe than that of embryos M/Z null for both APCs, this mutant
protein may not only block residual APC2 function, but may also interfere with
a parallel, APC2-independent means of nuclear retention.
Our data also provide insights into the domains of APC2 required for
nuclear retention, suggesting roles for the Arm repeats and the C terminus
(Fig. 10). Because
APC2S mutants exhibit much more
nuclear loss than do APC2N175K and
APC2c9, Arm repeat 5 may have a special importance,
perhaps by interfering with binding of a particular partner;
APC2S may also more profoundly
affect the overall structure of the Arm repeats.
APC structure and function in Wnt signaling
Our experiments provide an in vivo test of the function of proteins
truncated in the MCR (Fig.
10). APC2d40 and APC2g41
strongly reduce the ability to regulate Wg signaling, but are not as strong as
the null APC2g10, or APC2f90,
truncating APC2 at the end of the Arm repeats. A similar severely truncated
allele of mammalian APC led to higher levels of Wnt reporter activity in
cultured cells than did a truncation in the MCR
(Kielman et al., 2002). Our
data support the `just right' hypothesis
(Albuquerque et al., 2002),
which posits selection in tumors for mutations in which Wnt signaling is
elevated, but not too much. In this model, proteins truncated in the MCR
retain some ability to regulate ß-catenin, resulting in levels of Wnt
signaling that are above the threshold for polyp formation but not `too high',
which might be cell lethal. Our study is the first direct test of this
hypothesis using null alleles.
|
). However, because these cells retain the Arm
repeat-containing truncated protein found in the tumor, these two APC
fragments might exhibit intra-allelic complementation. Our data suggest that
the Arm repeats are crucial for full function of APC2 in the destruction
complex (Fig. 10). As our
missense mutations are not null for Wg regulation, APC proteins may retain
residual function in Wg signaling without the Arm repeats. Alternatively, the
mutations we isolated may not fully disrupt the repeats. Each Arm repeat
comprises three alpha-helices (e.g. Huber
et al., 1997). Together, the Arm repeats form a superhelical
structure with a large groove where partner proteins bind. Structure-based
sequence alignments (J. Stamos and W. Weis, personal communication) indicate
that four out of the five missense mutations (APC2e90,
APC2N175K APC2c9 and APC2b5)
are in helix 3 of Arm repeats 3, 6 and 7. These mutations affect residues
predicted to be in the protein core rather than those predicted to form the
binding surface. They may thus destabilize individual Arm repeats, or the Arm
repeat domain, without totally eliminating its function. This is consistent
with the temperature sensitivity of four out of the five Arm repeat
mutations.
APC2 and APC1 function redundantly in Wg signaling throughout
Drosophila development, despite differences in domain structure and
subcellular localization. This redundancy suggests that the shared domains -
the Arm repeats, the 15- and 20-amino acid repeats, the SAMP repeats and the
conserved sequences A and B - are sufficient for Wg regulation. We hypothesize
that the Arm repeats are the docking site for a binding partner important for
destruction-complex function. Using the temperature-sensitive allele
APC2S, we previously found that
the phenotype and the membrane association of the mutant protein varied in
parallel. Two of our weakest new alleles also exhibit residual membrane
association, thus the Arm repeats may bind a partner mediating cortical
localization of the destruction complex. However, this does not explain how
APC1 and APC2 can have different predominant localizations
(Akong et al., 2002a) and yet
be redundant. Perhaps low-level cortical accumulation of APC1, especially in
the absence of APC2, is sufficient for function. Future tests of this model
and identification of the relevant binding partner are needed. We will further
explore the function of the Arm repeats and other conserved regions as we
continue our analysis of this complex, multi-functional protein family.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/12/2407/DC1
* These authors contributed equally to this work 
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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
129,1751
-1762.
Akong, K., Grevengoed, E. E., Price, M. H., McCartney, B. M., Hayden, M. A., DeNofrio, J. C. and Peifer, M. (2002a). Drosophila APC2 and APC1 play overlapping roles in wingless signaling in the embryo and imaginal discs. Dev. Biol. 250,91 -100.[CrossRef][Medline]
Akong, K., McCartney, B. M. and Peifer, M. (2002b). Drosophila APC2 and APC1 have overlapping roles in the larval brain despite their distinct intracellular localizations. Dev. Biol. 250,71 -90.[CrossRef][Medline]
Albuquerque, C., Breukel, C., van der Luijt, R., Fidalgo, P.,
Lage, P., Slors, F. J., Leitao, C. N., Fodde, R. and Smits, R.
(2002). The `just-right' signaling model: APC somatic mutations
are selected based on a specific level of activation of the ß-catenin
signaling cascade. Hum. Mol. Genet.
11,1549
-1560.
Andreu, P., Colnot, S., Godard, C., Gad, S., Chafey, P.,
Niwa-Kawakita, M., Laurent-Puig, P., Kahn, A., Robine, S., Perret, C. et
al. (2005). Crypt-restricted proliferation and commitment to
the Paneth cell lineage following Apc loss in the mouse intestine.
Development 132,1443
-1451.
Chou, T. B. and Perrimon, N. (1996). The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144,1673 -1679.[Abstract]
Cliffe, A., Hamada, F. and Bienz, M. (2003). A role of Dishevelled in relocating axin to the plasma membrane during wingless signaling. Curr. Biol. 13,960 -966.[CrossRef][Medline]
Colnot, S., Decaens, T., Niwa-Kawakita, M., Godard, C., Hamard,
G., Kahn, A., Giovannini, M. and Perret, C. (2004).
Liver-targeted disruption of Apc in mice activates ß-catenin signaling
and leads to hepatocellular carcinomas. Proc. Natl. Acad. Sci.
USA 101,17216
-17221.
Cox, R. T., Kirkpatrick, C. and Peifer, M.
(1996). Armadillo is required for adherens junction assembly,
cell polarity, and morphogenesis during Drosophila embryogenesis.
J. Cell Biol. 134,133
-148.
Dikovskaya, D., Newton, I. P. and Nathke, I. S.
(2004). The adenomatous polyposis coli protein is required for
the formation of robust spindles formed in CSF Xenopus extracts.
Mol. Biol. Cell 15,2978
-2991.
Etienne-Manneville, S. and Hall, A. (2003). Cdc42 regulates GSK-3beta and adenomatous polyposis coli to control cell polarity. Nature 421,753 -756.[CrossRef][Medline]
Fodde, R., Kuipers, J., Rosenberg, C., Smits, R., Kielman, M., Gaspar, C., van Es, J. H., Breukel, C., Wiegant, J., Giles, R. H. et al. (2001). Mutations in the APC tumour suppressor gene cause chromosomal instability. Nat. Cell Biol. 3, 433-438.[CrossRef][Medline]
Foe, V. E., Odell, G. M. and Edgar, B. A. (1993). Mitosis and morphogenesis in the Drosophila embryo: point and counterpoint. In The Development of Drosophila, Vol. 1 (ed. M. Bate and A. Martinez-Arias), pp.149 -300. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Gaspar, C. and Fodde, R. (2004). APC dosage effects in tumorigenesis and stem cell differentiation. Int. J. Dev. Biol. 48,377 -386.[Medline]
Godt, D. and Tepass, U. (1998). Drosophila oocyte localization is mediated by differential cadherin-based adhesion. Nature 395,387 -391.[CrossRef][Medline]
Green, R. A. and Kaplan, K. B. (2003).
Chromosome instability in colorectal tumor cells is associated with defects in
microtubule plus-end attachments caused by a dominant mutation in APC.
J. Cell Biol. 163,949
-961.
Green, R. A., Wollman, R. and Kaplan, K. B.
(2005). APC and EB1 function together in mitosis to regulate
spindle dynamics and chromosome alignment. Mol. Biol.
Cell 16,4609
-4622.
Grigliatti, T. A. (1998). Mutagenesis. In Drosophila: A Practical Approach (ed. D. B. Roberts), pp. 55-84. Oxford: Oxford University Press.
Hamada, F. and Bienz, M. (2002). A Drosophila APC tumour suppressor homologue functions in cellular adhesion. Nat. Cell Biol. 4,208 -213.[CrossRef][Medline]
Hayashi, S., Rubinfeld, B., Souza, B., Polakis, P., Wieschaus,
E. and Levine, A. J. (1997). A Drosophila homolog of the
tumor suppressor gene adenomatous polyposis coli down-regulates ß-catenin
but its zygotic expression is not essential for the regulation of Armadillo.
Proc. Natl. Acad. Sci. USA
94,242
-247.
Huber, A. H., Nelson, W. J. and Weis, W. I. (1997). Three-dimensional structure of the armadillo repeat region of ß-catenin. Cell 90,871 -882.[CrossRef][Medline]
Kaplan, K. B., Burds, A. A., Swedlow, J. R., Bekir, S. S., Sorger, P. K. and Nathke, I. S. (2001). A role for the Adenomatous Polyposis Coli protein in chromosome segregation. Nat. Cell Biol. 3,429 -432.[CrossRef][Medline]
Kawasaki, Y., Sato, R. and Akiyama, T. (2003). Mutated APC and Asef are involved in the migration of colorectal tumour cells. Nat. Cell Biol. 5,211 -215.[CrossRef][Medline]
Kielman, M. F., Rindapaa, M., Gaspar, C., van Poppel, N., Breukel, C., van Leeuwen, S., Taketo, M. M., Roberts, S., Smits, R. and Fodde, R. (2002). Apc modulates embryonic stem-cell differentiation by controlling the dosage of ß-catenin signaling. Nat. Genet. 32,594 -605.[CrossRef][Medline]
Kim, K. M., Calabrese, P., Tavare, S. and Shibata, D. (2004). Enhanced stem cell survival in familial adenomatous polyposis. Am. J. Pathol. 164,1369 -1377.
