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First published online 9 July 2008
doi: 10.1242/dev.021766
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1 Department of Anatomy and Cell Biology, University of Melbourne, Parkville
3010, Melbourne, Australia.
2 Peter MacCallum Cancer Centre, East Melbourne 3002, Melbourne,
Australia.
* Author for correspondence (e-mail: l.quinn{at}unimelb.edu.au)
Accepted 12 June 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cell cycle, Drosophila, Wingless signalling
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and the Wnt signalling pathways
(Polakis, 2000) have been long
implicated in driving cell proliferation, which can lead to cancer initiation
and progression. In addition, steroid hormone signals can impact on Wnt
signalling to promote cancer progression
(Brisken et al., 2000;
Miller et al., 1998); however,
the genes important for connecting these pathways are unknown.
Drosophila melanogaster presents an ideal model for examining the
interplay between signalling pathways in cell proliferation control. In
particular, in the developing wing epithilelia of third instar larvae the Wg
pathway is important for the developmental cell cycle arrest at the
dorsal-ventral boundary in a region known as the zone of non-proliferating
cells (ZNC), which is essential for differentiation of cells along the wing
margin and formation of the wing blade
(Johnston and Edgar, 1998). In
the wing pouch, Wg expression is highest in the cell cycle arrested cells of
the ZNC, where it is required to downregulate the Drosophila
homologue of Myc transcription factor, dm, which drives growth and
G1- to S-phase progression in a functionally homologous manner to the
Myc oncogene (Johnston et al.,
1999). The Wg pathway also drives G2 arrest via downregulation of
the Cdc25 homologue stg
(Edgar and Datar, 1996), which
normally triggers mitotic entry by activating the Cdk1/Cyclin B kinase
(Edgar and Datar, 1996).
Wg-driven cell cycle exit in the ZNC, therefore, involves G1 arrest induced by
downregulation of dm (previously dmyc) and G2 arrest via
inhibition of stg.
Ecdysone (20-hydroxy ecdysone) is a key steroid hormone in
Drosophila, which has been predominantly studied for its role in the
remodelling of larval tissues, a requirement for patterning of adult
structures (Thummel, 1996).
During metamorphosis, a cascade of gene transcription is triggered by ecdysone
that activates dimerization of the ecdysone receptor (EcR), a member of the
nuclear receptor superfamily and its receptor binding partner Ultraspiricle
(USP) (Thummel, 1990;
Thummel, 1995;
Thummel, 1996). To date,
studies on the role of the ecdysone pathway in regulating cell cycle
progression have been limited. This is surprising as the ecdysone pulse is
essential for adult tissue morphogenesis, a process that requires coordination
of development with cell cycle, apoptosis and differentiation. Although
previous studies have revealed the molecular mechanism connecting ecdysone
signalling to the processes of apoptosis and differentiation (reviewed by
Baehrecke, 2000;
Jiang et al., 1997;
Yin and Thummel, 2005), there
are fewer studies showing connections between the ecdysone pulse and the
developmentally regulated cell cycles essential for generating adult tissues.
First, during larval development, ecdysone is required for morphogenetic
furrow progression in the eye imaginal disc
(Brennan et al., 1998) via the
early ecdysone response Broad-complex zinc-finger transcription factor Broad
(Br) (Brennan et al., 2001). In
addition, ectopic BR-C expression leads to ectopic endoreplication cycles
during oogenesis, suggesting that the ecdysone pathway can promote DNA
replication (Tzolovsky et al.,
1999). However, the molecular mechanism by which the ecdysone
response affects the cell proliferation machinery is unknown.
One target of ecdysone signalling is Crooked legs (crol),
which encodes a zinc-finger transcription factor
(D'Avino and Thummel, 1998).
Crol is upregulated by ecdysone during late larval/pre-pupal
development in imaginal discs, salivary glands and the CNS
(D'Avino and Thummel, 1998;
D'Avino and Thummel, 2000).
Pupal lethal crol mutants (crol4418) have defects
in ecdysone-induced gene expression
(D'Avino and Thummel, 1998) and
display abnormal wing development associated with reduced cell adhesion
(D'Avino and Thummel, 2000).
However, a role for Crol in regulating proliferation has not been
reported.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Fly
stocks were obtained from Bloomington, except en-GAL4, UAS-GFP and
Act<CD2<GAL4, UAS-GFP (Laura Johnston),
axinE77 (Jessica Treisman), stgAR2,
stg-lacZ line 6.4 (Bruce Edgar), PL35 (Allen Vincent),
dmP0 (Peter Gallant), armYD35 (Eric
Wieschaus), hsflp;arm-lacZ,M(2)z,FRT40A (Helen McNeill) and
PCNA-GFP and E2F91 (Bob Durunio).
Generation of clones in larval wing discs
For MARCM analysis, hsflp, UAS-GFP; FRT40A, gal80;
tubulin-GAL4 females were crossed to FRT40A lines for
control, crol[k05205] or crol[k05205]; UAS-p35
males. Heatshock was at 37°C for 1 hour, 72 hours after egg deposition
(aed) and larvae were aged to 120 hours aed prior to analysis. Alternatively,
heatshock was at 48 hours aed and larvae were analysed at 96 hours. To
generate clones in the Minute background, FRT40A, crol[k05205] males
were crossed to hsflp; arm-lacZ,M(2)z,FRT40A. For flip-out
clones Act<CD2<GAL4;UAS-GFP males were crossed to either
hsflp control, UAS-crol;UAS-p35 or hsflp:UAS-EcRDN
females and analysis was carried out for 120-hour larvae, 72 hours post
heatshock.
Antibody staining, BrdU and TUNEL labelling and microscopy
Crol antibody was generated to full-length Crol-GST fusion protein, in the
standard manner (Quinn et al.,
2001). Immunohistochemistry was carried out as previously
described (Quinn et al.,
2001). Antibodies used were: Wg (4D4) (Developmental Studies
Hybridoma Bank), Hfp (Trudi Schupbach), E(spl)m7 (Sarah Bray), Ci (2A1) (R. A.
Holmgren). Other antibodies used were anti-BrdU (Becton Dickinson) and
anti-phosphohistone H3 (Upstate Biotech.). All fluorescently labelled samples
were analysed by confocal microscopy (Zeiss LSM Meta).
Quantitative real-time PCR
mRNA was isolated from 20 sets of third instar larval heads either
overexpressing UAS-crol with Actin-GAL4 or from control
tissue (Actin-GAL4/+), and cDNA generated using the first-strand
synthesis kit (Invitrogen). wg primer sets for Q-RTPCR were
5'-gagatctccacaagcgaacc-3' and
5'-ccaatcacacggaagttgg-3'. Q-RTPCR was carried out with SYBR-GREEN
(Applied Biosystems) and the default conditions for ABI prism. Quantification
was achieved by normalization within each sample with cycle time for the GAPDH
primers (5'-ccgatgcgaccaaatccat-3' and
-5'agccatcacagtcgattc-3').
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)]
as a downstream target of Wg and demonstrated its cell cycle inhibitory
behaviour (Fig. 1A)
(Quinn et al., 2004). In order
to identify Hfp interactors, which would be potential regulators of cell
cycle, we carried out a genetic screen. Briefly, we screened deficiency and
mutant collections for dominant modifiers of a reduced wing phenotype that
results from overexpression of UAS-Hfp in the posterior compartment
(PC) of the larval wing disc, with the en-GAL4 driver
(Fig. 1C). As a strong enhancer
of the hfp overexpression phenotype, we identified the gene
Crooked legs (Crol) (Fig.
1D), which encodes a zinc-finger transcription factor
(D'Avino and Thummel, 1998).
Crol has been shown to be normally required for wing development; however,
expression from the crol4418-lacZ enhancer trap was not detected in
the wing disc (D'Avino and Thummel,
1998). In order to determine whether Crol is normally expressed in
the wing, we stained wild-type wing discs with an anti-Crol antibody. Crol
protein was detected in the nucleus of most wing disc cells from both the
pouch and the hinge (Fig.
1E,F). Co-staining with Wg
(Fig. 1G) revealed a decrease
in Crol expression within the Wg-expressing cells comprising the ZNC, when
compared with cells flanking the ZNC. We demonstrated the specificity of the
Crol antibody by showing increased Crol protein in flip-out clones
over-expressing the UAS-crol transgene
(Fig. 1H,I). The detection of
Crol in the pouch and hinge is consistent with Crol being required to regulate
transcription during wing development. We therefore sought to test whether
enhancement of the hfp overexpression phenotype was due to Crol
normally being required for cell cycle in the wing.
Crol is required for cell cycle progression
To determine whether Crol was required for cell cycle in the wing disc, we
generated mutant clones for the embryonic lethal crol mutant
(crolk05205) in third instar larvae, using MARCM
(Lee et al., 2000). Wing discs
from 120 hour larvae contained very few crol-/- clones in
the pouch (Fig. 2C,D), which
differentiates into the wing blade, compared with wild type
(Fig. 2A,B). By contrast,
crol mutant clones were observed in imaginal tissue destined to give
rise to hinge (Fig. 2C,D). As
both control and crol mutant clones are induced at the same
developmental time point [heat shock induced Flp-mediated recombination at 72
hours after egg deposition (aed)], this result suggests that
crol-/- cells within the wing pouch, but not in the hinge
region, either fail to proliferate and/or are removed by apoptosis. Although
previous analysis had verified that crolk05205 contained a
single P element in the crol promoter
(Spradling et al., 1999), we
wanted to show that the phenotype was due to loss of Crol function. Indeed,
expression of the UAS-crol transgene in
crolk05205 mutant clones resulted in rescue of clonal size
and survival (Fig. 2E). Thus,
the reduced size of the clonal tissue within the pouch is due to loss of
crol.
|
). In the p35
background, more crol mutant cells were observed
(Fig. 2J), suggesting that
crol mutant tissue is normally removed by apoptosis. It is important
to note, however, that inhibition of caspase-dependent death does not
completely restore the size and number of crol mutant clones (compare
Fig. 2J with the control in
Fig. 2A), suggesting that
apoptosis is not the sole explanation for the reduction in size and number of
crol mutant clones.
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Ectopic overexpression of Crol drives cell cycle progression
To determine whether Crol might drive proliferation, we overexpressed
UAS-crol in the posterior compartment (PC) of the wing disc with
en-GAL4. Increased S phases were evident in the PC of the wing pouch
ZNC in cells over-expressing UAS-crol
(Fig. 3C,D) compared with the
control (Fig. 3A,B). In
addition, PH3 staining of the same disc revealed mitotic cells within the PC
ZNC (Fig. 3G,H) compared with
the control (Fig. 3E,F). As
cells within the PC ZNC have normally exited the cell cycle and are arrested
in G1 phase prior to differentiation, this suggests that overexpression of
crol interferes with developmentally regulated cell cycle exit in the
wing pouch.
|
).
Indeed, TUNEL labelling revealed massive levels of apoptosis within
crol-expressing cells of the PC
(Fig. 3I,J). Although we have
shown that overexpression of crol causes wing imaginal cells to die
by apoptosis, it is unlikely that the primary role of Crol is to drive
apoptosis, as cells lacking crol have decreased cell cycles and
increased cell death rather than enhanced survival
(Fig. 2). Therefore the
increased apoptosis in cells overexpressing crol is most probably a
consequence of ectopic proliferation, rather than a direct affect of
crol in driving cell death.
Overproliferation results when apoptosis is blocked in cells overexpressing crol
The co-expression of UAS-p35 to inhibit caspase-dependent
apoptosis in crol-expressing cells revealed the contribution of
apoptosis to the reduced PC. Indeed, we found a substantial increase in the
size of the PC from 120-hour larvae overexpressing UAS-crol in the
presence of UAS-p35 (Fig.
3L compared with control Fig.
3K), which was associated with a massive increase in S phase
(Fig. 3M,N) and mitosis
(Fig. 3O,P). Analysis of
96-hour wing discs co-expressing UAS-crol and UAS-p35 also
revealed an increase in mitotic cells (see Fig. S1A-C in the supplementary
material) and S phases in the PC (see Fig. S1D-F in the supplementary
material). Importantly, crol overexpression results in ectopic
mitoses (see Fig. S1G-I in the supplementary material) and S phase cells (see
Fig. S1J-L in the supplementary material) within the PC ZNC. Thus,
crol over-expression can override the developmental signalling that
promotes cell cycle exit in the ZNC. In order to further characterise the
overgrowth, we carried out cell cycle analysis of flip-out clones. As found
for wing discs co-expressing crol and p35 in the PC, discs containing
clones co-expressing UAS-crol and UAS-p35 were also
overgrown and clonal tissue had increased BrdU
(Fig. 3R,S, compare with
control in Fig. 3Q) and PH3
(Fig. 3V,W, compared with the
control in Fig. 3U).
Quantification revealed a significant increase in both BrdU
(Fig. 3T; P<0.0001)
and PH3 (Fig. 3X;
P<0.0033) from equivalent clone areas for
UAS-crol;UAS-p35 clones compared with UAS-p35
alone.
Collectively, these data (Figs 2 and 3) suggest that Crol is necessary for normal cell cycle progression, and overexpression of crol can drive the cell cycle in the larval wing disc, in a manner that overcomes developmental cell cycle inhibition. Furthermore, the accelerated rate of cell cycle progression in tissue overexpressing crol results in a high degree of compensatory apoptosis that, if blocked, leads to massive tissue overgrowth.
Interestingly, in the p35 background, some cells in the PC lose expression
of GFP, which is first evident as small patches of non-GFP cells at 96 hours
(see Fig. S1A-F in the supplementary material) and more striking at 120 hours
(see Fig. S1G-L in the supplementary material). Increased proliferation is
observed in both the GFP-positive cells still overexpressing crol and
in the patch of cells that have lost GFP. Although it is unclear why patches
of cells lose GFP, staining with anti-En revealed continued expression of En,
suggesting that these cells maintain their posterior identity (see Fig. S1M-O
in the supplementary material). It is possible these cells have acquired an
`undead' state owing to the expression of p35, as caspase-inhibited
cells initiate apoptosis but do not die. `Undead' cells can cause additional
proliferation of surrounding cells owing to transient signals sent from the
`undead' cell (Huh et al.,
2004; Perez-Garijo et al.,
2004; Ryoo et al.,
2004). Thus, it is possible that cells co-expressing crol
and p35 might exhibit `undead' properties and affect secreted
signalling molecules to cause non-autonomous affects on the surrounding cells.
Indeed, increased proliferation is observed in cells adjacent to clones
co-expressing UAS-crol and UAS-p35
(Fig. 3R,S,V,W). Importantly,
expression of p35 alone in the PC does not affect proliferation (see Fig. S2
in the supplementary material). Thus, crol overexpression drives
proliferation, but additional cells are normally removed by compensatory
apoptosis (Reis and Edgar,
2004). If apoptosis is blocked with p35, these cells may continue
to proliferate or acquire an `undead state'. Therefore, expression of p35 in
cells overexpressing crol results in tissue overgrowth because (1)
extra cells resulting from overproliferation are not removed by compensatory
apoptosis and (2) the potentially `undead' cells can promote further growth by
sending growth promoting signals to neighbouring cells.
The crol overexpression phenotype is sensitive to the dose of cell cycle regulators
Thus far we have shown that loss of crol results in reduced cell
cycles and that overexpression of crol leads to ectopic
proliferation. To investigate whether the effects of increased Crol were
sensitive to cell cycle regulators, we used the adult wing phenotype resulting
from crol overexpression. The crol overexpression adult wing
phenotype represents the effects of crol on proliferation and on cell
death seen in larval wing disc development
(Fig. 3), and results in a
reduced PC (Fig. 4A). To
examine the interaction between cell growth/cycle genes and crol, we
tested whether various cell cycle mutants could modify the crol
overexpression phenotype. Halving the dose of dm
(Johnston et al., 1999)
suppressed the crol phenotype
(Fig. 4B), suggesting
dm is required downstream of Crol and is rate-limiting for
Crol-induced G1- to S-phase progression. This was not specific to dm,
suppression of the crol phenotype was also achieved by halving the
dose of the essential S-phase Cyclin cycE
(Knoblich et al., 1994)
(Fig. 4C). Suppression of the
crol phenotype also resulted from halving the dose of either
stg/Cdc25 (Edgar and Datar,
1996) or the G2-M Cyclin CycB
(Lehner and O'Farrell, 1990)
(Fig. 4D,E, respectively).
Thus, the crol overexpression phenotype is also sensitive to the dose
of G2-M regulators, in accordance with our finding that overexpression of
crol increases mitoses. Consistent with the interaction of both G1-S
and G2-M regulators with the crol overexpression phenotype, we found
suppression by halving the dose of the E2F1 transcription factor
(Fig. 4F), which upregulates
both cycE and stg to drive cells through both S phase and
mitosis (Reis and Edgar,
2004).
Crol upregulates dm, stg and E2F1-dependent gene transcription
In order to test whether Crol was driving proliferation via upregulating
transcription of essential cell cycle genes, we used lacZ enhancer
traps for dm (PL35)
(Bourbon et al., 2002) and
stg (stg-lacZ 6.4)
(Lehman et al., 1999).
Although we found that β-gal staining for
PL35-dm-lacZ/+ and stg-lacZ/+ was barely above
background (data not shown), when we overexpressed UAS-crol with
en-GAL4 in the UAS-p35 background, increased
dm-lacZ staining was observed in the PC of the wing disc
(Fig. 4G-I). In addition, using
ptc-GAL4 to co-express UAS-crol and UAS-p35 in the
stg-lacZ reporter background showed increased β-gal expression
in the Ptc domain (Fig. 4J-L).
These findings are consistent with overexpression of crol resulting
in upregulation of dm transcription to drive S-phase progression and
of stg transcription to drive M-phase progression.
To determine whether overexpression of crol affects the
E2F1 transcription factor, we used a GFP reporter for E2F1
transcriptional activity [the proliferating cell nuclear antigen GFP reporter,
PCNA-GFP (Thacker et al.,
2003)]. We observed a massive increase in PCNA-GFP staining across
the entire PC of the wing discs expressing UAS-crol with
en-GAL4 (Fig. 4N,O)
compared with the control (Fig.
4M), showing Crol acts to upregulate E2F transcriptional
activity.
Crol blocks the Wg signalling pathway required for inhibition of cell proliferation
The finding that crol overexpression drives ZNC cells through the
cell cycle combined with the knowledge that Wg signalling is important for
cell cycle arrest in the ZNC (Johnston and
Edgar, 1998) suggested Crol might act to block the Wg pathway. To
determine whether Crol might mediate cell cycle via affects on the Wg pathway,
we tested for dominant modification of the crol overexpression wing
phenotype by Wg pathway mutants (Fig.
5). We found that halving the dose of genes required for Wg
activation, such as the β-catenin homologue armadillo
(arm) (Tolwinski and Wieschaus,
2001), resulted in enhancement of the crol overexpression
phenotype (Fig. 5B).
Conversely, halving the dose of inhibitory components of the Wg pathway, such
as Axin (Willert et al.,
1999) or Sgg/GSK3β
(Siegfried et al., 1992),
resulted in suppression of the wing phenotype
(Fig. 5C,D, respectively). The
crol overexpression phenotype is therefore sensitive to the dose of
Wg pathway components, in a manner consistent with Crol acting to antagonise
Wg signalling in order to overcome cell cycle inhibitory effects of Wg in the
wing pouch.
Reducing Wg signalling restores crol-/- clonal size
To determine whether the failure to proliferate in crol mutant
clones was dependent on increased Wg signalling within the clone, we tested
whether the growth deficiency could be suppressed by halving the dose of
arm. Analysis of 120-hour third instar wing discs revealed that
growth of crol mutant clones was restored
(Fig. 5E-G) compared with the
crol mutant alone (Fig.
2C,D). Thus, the proliferative disadvantage of the crol
mutant clones is sensitive to the dose of the Wg pathway transcription factor
Arm, suggesting that failure of the crol mutant clones to proliferate
is dependent on signalling through the Wg pathway.
Crol downregulates wg transcription and is essential for suppression of Wg
To determine whether crol acts to repress Wg, we first tested
whether overexpression of UAS-crol in the PC with en-GAL4
affected wg-lacZ enhancer-trap activity
(Fig. 5H-M). The level of
β-gal staining was reduced in the PC cells overexpressing crol
when compared with either the AC of the same disc
(Fig. 5K-M) or the PC from the
control (Fig. 5H-J). We
counterstained with DAPI to show that the reduced staining was not due to the
loss of cells via apoptosis, as we observe abundant cells in the apical region
of the disc (Fig. 5M). This
suggests that wg promoter activity is downregulated in response to
crol overexpression in the wing pouch.
Although these findings are consistent with Crol behaving as a repressor of wg gene transcription, we wanted to determine whether Crol was necessary for downregulation of wg. As we had previously found that crol mutant cells proliferated more slowly than surrounding wild-type tissue, we attempted to ameliorate this proliferative disadvantage by generating crol clones in a minute [M(Z)2] background. Although homozygous crol clones were eliminated from the pouch in a wild-type background, clones induced at the equivalent stage in the slow-growing Minute background were detected in the pouch (Fig. 5Q-S). Nevertheless, control clones induced at the same time were much larger (Fig. 5N-P), suggesting that crol-/- cells grow much more slowly than Minute heterozygous cells. In particular, we noticed that the only crol-/- clones comprising more than one or two cells were adjacent to the endogenous Wg domain, from either the pouch or the hinge (Fig. 5Q-S). However, regardless of the position of the crol mutant clone, the level of Wg protein was elevated (Fig. 5Q-S) when compared with control clones (Fig. 5N-P); Wg protein associated with the Wg domains in the ZNC (Fig. 5T-V) or in the hinge (Fig. 5W-Y) was expanded and ectopic Wg expression was seen in the small one or two cell clones isolated from endogenous Wg domains (Fig. 5W-Y). In summary, these data suggest that Crol is normally required for repression of Wg expression.
|
Crol acts to block Hfp expression in the wing disc
Our previous studies have demonstrated that Hfp is upregulated in response
to Wg signalling and is required for cell cycle arrest in the ZNC
(Quinn et al., 2004). Hfp
negatively regulates G1- to S-phase progression by downregulating dm
and inhibits mitosis via Stg. We therefore tested whether increased
proliferation driven by Crol was associated with changes to Hfp protein levels
(Fig. 6). Hfp protein is
normally detected in the nuclei of all wing imaginal disc cells
(Fig. 6A-C); however, a clear
reduction in Hfp was observed in PC cells expressing UAS-crol
(Fig. 6D-F). Thus, Crol leads
to downregulation of Hfp. Given this result, the mechanism by which the
crol mutant dominantly enhances the hfp overexpression
phenotype (Fig. 1) may be via
its effect on increasing Wg levels, which would in turn result in increased
Hfp levels and further cell cycle inhibition.
Wg regulation is not achieved via indirect affects on the Hh or Notch pathways
The Hedgehog (Hh) and Notch (N) pathways are key upstream regulators of Wg
in the wing disc. To determine whether Crol might regulate wg
indirectly, via affects on the Hh or N pathways, we tested whether these
pathways were altered in crol mutant clones or tissue overexpressing
crol.
The zinc-finger transcription factor Cubitus interruptus (Ci) is the
mediator of Hh-dependent transcriptional activation of wg. Ci is both
necessary and sufficient to drive expression of Hh-responsive genes, including
the upregulation of wg transcription
(Aza-Blanc and Kornberg, 1999).
Upregulation of wg could therefore occur via Hh signalling. However,
ectopic Ci protein was not detected in crol mutant clones, suggesting
that Crol does not affect wg transcription indirectly via the Hh
pathway (see Fig. S3A-C in the supplementary material).
Notch activity also plays a role in cell cycle arrest during wing
development (Johnston and Edgar,
1998). Notch is activated in cells along the dorsoventral (DV)
boundary (ZNC) of the wing disc, where it is required for Wg expression
(de Celis et al., 1996).
However, decreased levels of the Notch target, En(spl)m7 were not observed in
crol overexpressing cells, suggesting Notch signalling is not
downregulated by Crol (see Fig. S3D-F in the supplementary material). Taken
together, these results suggest that Crol does not affect wg
transcription indirectly via effects on the Notch or Hh pathways.
|
|
)]
(Fig. 7). In the
wg-lacZ background, we observed increased levels of β-gal
staining within clones adjacent to the Wg domain in both the pouch and the
hinge (Fig. 7A,B). These
findings are consistent with the EcR normally being required to inhibit
wg transcription in the wing imaginal disc. As predicted, the
increased level of Wg was associated with a significant reduction in cell
cycle progression in UAS-EcRdN clones, measured by BrdU
(Fig. 7C, quantified in
7D, P<0.0001) and
PH3 (Fig. 7E, quantified in
Fig. 7F, P<0.0001).
These results are consistent with EcR normally being required for repression
of Wg and therefore cell cycle progression in the wing pouch. However, unlike
crol-/- clones, UAS-EcRdN clones are not
out-competed and eliminated by apoptosis, which might be a consequence of
reduced levels of the cell death targets normally activated by the EcR pathway
(reviewed by Baehrecke, 2000;
Jiang et al., 1997;
Yin and Thummel, 2005).
Therefore, in contrast to loss of Crol function, blocking the ecdysone pathway
upstream of Crol results in survival. Thus, in this background, the increase
in wg expression is associated with decreased proliferation, but not
with reduced survival. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Wg signal within the wing imaginal disc differentially affects
proliferation. In the pouch, Wg drives cell cycle exit and differentiation,
while upregulation of Wg signalling in the hinge drives proliferation
(Johnston and Sanders, 2003).
In accordance with Crol being required to repress Wg throughout the wing disc,
Crol protein is present in both the pouch and hinge, and loss of Crol results
in increased Wg in both domains. In agreement with the proliferation driven by
Crol being mediated by the Wg signal, we observe differential affects on
proliferation for crol mutant clones in the pouch compared with the
hinge. We observe frequent crol-/- clones in the hinge,
most probably as a consequence of Wg promoting proliferation in these cells
(Johnston and Sanders, 2003).
Consistent with Wg repressing proliferation in the pouch, we see very few
crol-/- clones in this region.
However, if the only function of Crol was to inhibit wg
transcription, it would be expected that loss-of-function crol clones
would behave similarly to clones with increased Wg in regard to proliferation
and survival. However, although Wg promotes survival
(Johnston and Sanders, 2003),
crol-/- clones are removed by apoptosis, which is most
probably due to cell competition (de la
Cova et al., 2004). As Crol has also been shown to be required for
integrin expression, removal of crol-/- cells from the
wing epithelium may also be a consequence of a potential reduction in cell
adhesion (D'Avino and Thummel,
2000). Indeed, loss of integrin-mediated cell adhesion to the
extracellular matrix (ECM) has been shown to cause apoptosis, a process known
as anoikis (Jan et al., 2004).
Thus, in crol-/- clones, increased Wg leads to reduced
proliferation, but reductions to integrin levels and cell adhesion would be
expected to reduce survival.
Our current working model for how Crol connects steroid hormone signalling
to cell cycle progression is shown in Fig.
7. First, Crol is upregulated in response to ecdysone
(D'Avino and Thummel, 1998) and
then the increased level of Crol results in decreased wg
transcription. Reduced Wg signalling results in downregulation of Hfp, which
results in increased dm expression and S-phase progression
(Quinn et al., 2004).
Increased Dm levels also lead to upregulation of its cell cycle targets
cycE, cycD and cdk4, resulting in inactivation of
Rbf and increased activity of the S phase transcription factor E2f1
(Duman-Scheel et al., 2004).
Hfp downregulation also leads to increased mitosis owing to increased Stg
protein levels (Quinn et al.,
2004). Thus, we have uncovered a novel mechanism for cell cycle
regulation, whereby Crol acts to link steroid hormone signals to the Wg
pathway and the regulation of crucial cell cycle targets.
Our finding that the early response ecdysone pathway target Crol is crucial
for developmental cell cycle progression adds significantly to previous
studies, which have made connections between the ecdysone pulse and
developmentally regulated apoptosis and differentiation. Our finding that the
ecdysone-inducible gene crol is required for cell cycle progression,
provides evidence that the Ecdysone pulse is essential for all aspects of
adult tissue morphogenesis, being required for coordination of developmental
progression with cell cycle, apoptosis and differentiation. Of particular
developmental importance, crol induction by the ecdysone pulse at the
larval-pupal transition (Thummel,
1996), may be crucial for driving the final rounds of
proliferation of the epithelial cells of the pupal wing blade prior to their
final exit from the cell cycle between 20 and 24 hours after pupariation,
before they undergo terminal differentiation
(Buttitta et al., 2007;
Milan et al., 1996).
Connections between mammalian steroid hormone pathways and regulation of
cell cycle genes are associated with a variety of hormone-dependent diseases,
including cancer (Nilsson et al.,
2004). In murine embryonic stem (ES) cells, oestrogen has been
shown to promote proliferation associated with increased mRNA expression of
proto-oncogenes, including Myc
(Han et al., 2006). Our work
suggests that steroid hormone regulation of proliferation via the Myc family
is conserved between flies and humans. However, whether mammalian steroid
hormone signalling pathways regulate the human homologue of Crol (called
ZNF84) to affect wg/Wnt transcription, hfp/Fir levels and
cell cycle progression remains to be determined.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/16/2707/DC1
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ACKNOWLEDGMENTS |
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