1 Trescowthick Research Laboratories, Peter MacCallum Cancer Centre, St Andrew's
Place, East Melbourne, VIC 3002 Australia
2 ARC Centre of Excellence in Biotechnology and Development, University of
Melbourne
* Authors for correspondence (e-mail: david.bowtell{at}petermac.org and helena.richardson{at}petermac.org)
Accepted 27 November 2003
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SUMMARY |
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Key words: Drosophila, Hfp, Cell proliferation
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Introduction |
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FIR and its Drosophila orthologue Hfp have an evolutionarily
conserved function in pre-mRNA splicing. Mammalian FIR was originally isolated
as poly(U) binding splicing factor (PUF60), and together with the splicing
factors p54 and U2AF, promotes RNA splicing in vitro
(Page-McCaw et al., 1999).
Furthermore, FIR directly interacts with U2AF65, the large subunit of U2AF
(Poleev et al., 2000
). FIR and
U2AF65 have similar domain structures, including the multiple RNA-recognition
motif (RRM) domains. The Drosophila homologue of FIR has a conserved
role in regulating pre-mRNA splicing and is known as Half pint (Hfp)
(Van Buskirk and Schupbach,
2002
), dPUF68 (Page-McCaw et
al., 1999
) or pUbsf
(http://flybase.bio.indiana.edu/).
Hfp controls RNA splicing of several Drosophila ovarian genes,
including ovarian tumor (otu)
(Van Buskirk and Schupbach,
2002
) (reviewed by Rio,
2002
). The hfp mutant ovary phenotype, which includes
defective germline proliferation that results in reduced numbers of germline
cells per egg chamber, is rescued by re-expressing an appropriately spliced
otu isoform (Van Buskirk and
Schupbach, 2002
). Therefore Drosophila Hfp, like its
mammalian counterpart FIR, has an important role in tissue-specific regulation
of alternative splicing.
In addition to regulating splicing of pre-mRNA, mammalian FIR is also an
important regulator of Myc gene activity
(Eisenman, 2001;
Liu et al., 2000
). Expression
of Myc is tightly regulated, at the level of transcription,
translation and protein stability
(Eisenman, 2001
). One
mechanism for control of Myc transcriptional initiation and
elongation is mediated by the far upstream element (FUSE), a DNA sequence
located 1500 bp upstream of the Myc promoter. The FUSE binding
protein (FBP), a KH domain transcriptional activator, binds the FUSE and is
absolutely required for Myc expression and cell growth in mammalian
cells (Duncan et al., 1994
;
He et al., 2000
). The FBP
interacting repressor (FIR) counteracts FBP function by forming a ternary
complex with FBP and the FUSE to repress Myc transcription
(Liu et al., 2000
). The
N-terminal repression domain of FIR interacts with the basal transcription
component TFIIH and interferes with promoter clearance. The in vivo importance
of this mechanism is not clear; however, mutations in ERCC2 or
ERCC3 (which encode TFIIH subunits corresponding to the xeroderma
pigmentosum (XP) complementation groups XPD and
XPB, respectively) impair regulation of Myc expression by
FBP and FIR. This may contribute to cancer risk in individuals with XP
mutations (Liu et al.,
2001
).
The proteins encoded by the myc family of proto-oncogenes are
important regulators of cell growth (size and mass increase), proliferation,
differentiation and apoptosis (Eisenman,
2001). In response to mitogenic signalling, Myc can inhibit
differentiation and either promotes cell growth and proliferation or
apoptosis, depending on the context. Myc proteins form stable heterodimers
with Max proteins to modulate expression of target genes by binding E box DNA
sequences. Although primarily a transcriptional activator, Myc can also
inhibit the expression of certain target genes. Deregulated Myc expression is
potently oncogenic and is one of the most frequently observed molecular
abnormalities in human cancers. Despite this, regulation of Myc expression and
its role in tumourigenesis has not been clearly defined.
The Drosophila dmyc (dm - FlyBase) and dmax
(Max - FlyBase) gene products also form heterodimers, bind E-box DNA
sequences and activate transcription
(Eisenman, 2001;
Gallant et al., 1996
). Gain
and loss of function studies in Drosophila have revealed that the
primary in vivo function of dMyc is to stimulate cell growth. dmyc
mutations cause cellular growth retardation; resulting in small flies with
small cells (Gallant et al.,
1996
; Johnston and Edgar,
1998
; Johnston et al.,
1999
; Schreiber-Agus et al.,
1997
). Conversely, overexpression of dmyc in the wing
imaginal disc promotes cell growth, leading to increased cell size
(Johnston et al., 1999
).
Although dMyc-induced cell growth is accompanied by faster G1/S phase
progression, the overall cell division rate of dmyc overexpressing
cells remains normal due to an extended G2 phase
(Johnston et al., 1999
), which
arises because the Drosophila homologue of Cdc25 phosphatase, String
(Stg), is rate limiting for G2-M cell cycle progression
(Edgar and O'Farrell, 1989
;
Edgar and O'Farrell, 1990
).
Stg triggers mitotic entry by dephosphorylating, and thereby activating the
Cdk1/Cyclin B kinase (Edgar et al.,
1994
).
The Wingless-signalling pathway regulates both dmyc and
stg expression during Drosophila wing development. During
third instar larval development, the dorsoventral compartment boundary of the
wing imaginal disc forms a zone of cells arrested in G1 or G2, termed the ZNC.
A band of Wingless (Wg) expression controls cell cycle arrest within the ZNC
(Johnston and Edgar, 1998).
While dmyc is expressed in proliferating zones within the wing,
expression is normally low in the ZNC, and ectopic expression of dmyc
in the ZNC prevents cell cycle exit
(Johnston et al., 1999
).
Furthermore, inhibition of Wg signalling in the ZNC, via expression of
dominant negative TCF, results in ectopic dmyc expression in the ZNC
(Johnston et al., 1999
). These
studies show that Wg signalling represses dmyc expression within the
ZNC; however, whether the repression of dmyc transcription by TCF is
direct or indirect is unknown. While the posterior region of the ZNC is
comprised solely of G1-arrested cells, the anterior compartment of the ZNC
contains a band of G1-arrested cells at the dorsoventral boundary that is
sandwiched between anterior-dorsal and anterior-ventral G2-arrested domains.
Wg signalling is required for the downregulation of stg and
associated G2-arrest in the anterior of the ZNC. This occurs indirectly, via
Wg upregulating achaete and scute, which in turn
downregulate stg, resulting in the G2-arrested cells in the ZNC
(Johnston and Edgar, 1998
).
Whether Achaete and Scute act directly on the stg promoter is
unknown.
Here we describe an alternative role for Hfp, as a negative regulator of cell cycle progression in Drosophila imaginal tissues. We show that within the ZNC of hypomorphic hfp mutant wing discs, cells undergo ectopic S phases, suggesting that, like FIR, Hfp might control cell proliferation by regulating dmyc expression. Indeed, elevated dmyc expression was detected in hfp mutant clones, and reducing the dosage of hfp rescued the dmyc mutant ovary phenotype. Unlike dmyc overexpression, hfp mutants did not affect cell growth, although cell proliferation was increased. This can be explained via an affect of Hfp on the G2-M phase transition, since hfp mutants can rescue the cycle 14 G2-arrest phenotype of an stg mutant. Furthermore, Hfp protein was elevated in response to Wg pathway signalling. Taken together, these results are consistent with Hfp playing an important role in cell cycle arrest downstream of Wg signalling. These findings suggest that Hfp links patterning signals to cell growth and proliferation in the Drosophila wing.
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Materials and methods |
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In-situ hybridization, antibody staining, BrdU and TUNEL labelling and microscopy
mRNA in-situ hybridization was carried out as described in previous methods
(Dorstyn et al., 1999) except
the signal was detected using fast-red substrate (Roche). Following in-situ
hybridization, clones were distinguished using a rabbit anti-GFP polyclonal
antibody (Molecular Probes), detected using an anti-rabbit-biotin conjugated
secondary antibody, followed by streptavidin-Alexa488 (Molecular Probes).
After in-situ hybridization of ovaries, DNA staining was carried out with
Oligreen (Molecular Probes) to assist with staging.
Immunohistochemistry, including TUNEL and BrdU labelling of
Drosophila larval tissues and embryos, was carried out as previously
described unless otherwise indicated
(Quinn et al., 2000;
Quinn et al., 2001
). The
monoclonal Hfp antibody (Trudi Schupbach) was detected using an
anti-mouse-biotin conjugated secondary antibody followed by
streptavidin-lissamine rhodamine (Jackson). TUNEL staining was carried out
using the in-situ cell death detection kit TRred (Roche). Other antibodies
used were anti-BrdU monoclonal antibody (Becton Dickinson) and rabbit
anti-phosphohistone H3 (Santa Cruz), rabbit anti-GFP (Molecular Probes),
rabbit anti-Cyclin B (David Glover), rat anti-Geminin
(Quinn et al., 2001
), rabbit
anti-ßgal (Rockland) and rabbit anti-Stg (Bruce Edgar). Ovaries were
stained with phalloidin-rhodamine, 0.1% in PBT for 1 hour (Sigma), prior to
staining with Oligreen (Molecular Probes). All fluorescently labelled samples
were analysed by confocal microscopy (Biorad MRC1000). Scanning electron
micrographs of adult eyes were generated as previously described using a Field
Emission Scanning Electron Microscope
(Secombe et al., 1998
).
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Results |
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In order to investigate whether Hfp regulates cell proliferation during
Drosophila development, we measured BrdU incorporation in wing discs
from wandering hfpEP/hfpEP larvae. In
wild-type wing discs the ZNC is clearly marked by the absence of BrdU
labelling (Fig. 1I). The number
of S-phase cells was markedly increased in hfpEP mutant
wing discs, BrdU incorporation was uniform across the disc and cell cycle
arrest was not evident in the ZNC region
(Fig. 1L). Strikingly,
anti-phosphohistone H3 antibody staining of mitotic cells
(Hans and Dimitrov, 2001), was
also elevated, indicating an overall increase in cell proliferation in
hfp wing discs (Fig.
1M; 127±7 mitotic cells per disc) compared with wild type
(Fig. 1J; 75±8 mitotic
cells; n=5 discs, P<0.01).
The developing eye is a sensitive system for analysis of cell proliferation. During wild-type eye development, a wave of differentiation moves from posterior to anterior across the third instar eye imaginal disc. Within the morphogenetic furrow (MF) cells are arrested in G1 and posterior to the MF a subset of cells enter a synchronous S phase (Fig. 1O) while other cells begin differentiation to form ommatidial pre-clusters, followed by a band of mitotic cells known as the second mitotic wave (Fig. 1P). Analysis of the band of S phases posterior of the MF (Fig. 1R) and the second mitotic wave (Fig. 1S) in hfp mutant eye discs revealed that the S-phase band is generally broader than for wild type, but the second mitotic wave does not occur prematurely, suggesting that Hfp might normally be required for the pre-cluster cells to cease division.
Despite increased proliferation in hfpEP mutant discs, they were not overgrown compared with wild type (data not shown). We did not observe an obvious difference in cell size between hfpEP mutant wing disc cells by either cross section (Fig. 1V compared with wild type, Fig. 1U) or by transverse section (data not shown), suggesting that increased cell death may accompany increased proliferation in this tissue to account for the fact that the discs are similar in size to wild type. Indeed, TUNEL staining revealed an increase in the number of apoptotic cells in the wing imaginal discs of hfp mutants (Fig. 1X; 143±17 apoptotic cells per disc) compared with wild-type larvae (Fig. 1W; 26±9 apoptotic cells, n=5 discs, P<0.005).
Therefore, although increased cell cycles were observed in hfp
mutant wing discs, the overall disc size was similar to wild type, as ectopic
proliferation was apparently balanced by increased apoptosis. The elevated
cell death observed in hfp mutant wing discs is likely to be a
secondary consequence of deregulated cell proliferation. In
Drosophila, compensatory cell death in the face of hyperproliferation
appears to be a general mechanism for maintaining normal compartment size and
is also observed in imaginal discs upon ectopic expression of dmyc
(Johnston et al., 1999), the
cell cycle transcription factor E2F (Asano
et al., 1996
) or both the G1-S phase regulator Cyclin E and the
G2-M phase regulator Cdc25/Stg (Neufeld et
al., 1998
).
Hfp overexpression inhibits cell cycle entry
The observation that loss of Hfp promotes cell cycle entry prompted us to
examine whether overexpression of Hfp could block cell proliferation. We
generated transgenic flies containing a UAS-hfp transgene in order to
ectopically express Hfp using various GAL4 drivers (Brand and
Perrimon, 1993). Ubiquitous expression of Hfp using armadillo-GAL4
(arm-GAL4) partially rescued the pupal lethality of
hfpEP/hfpEP animals, verifying
transgene function (data not shown). We specifically overexpressed
hfp in cells posterior to the MF in the eye disc using the
GMR-GAL4 driver. Expression of two copies of UAS-hfp under
control of GMR-GAL4 (GMR-GAL4,UAS-hfp/+; UAS-hfp/+)
resulted in flies with disorganized adult eyes that were slightly smaller than
wild type (Fig. 2B). Third
instar eye discs from GMR-GAL4,UAS-hfp/+; UAS-hfp/+ larvae
showed reduced BrdU incorporation in the S-phase band posterior to the MF
(Fig. 2D) compared with wild
type (Fig. 2C). In addition,
reduced numbers of cells staining with anti-phosphohistone H3 were observed
posterior to the S-phase band of GMR-GAL4,UAS-hfp/+;
UAS-hfp/+ eye discs (Fig.
2F) compared with wild type
(Fig. 2E). Thus overexpression
of hfp can inhibit S phase entry and mitoses posterior to the MF,
consistent with a role for Hfp in negatively regulating cell cycle
progression.
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Loss of hfp suppresses the cell cycle inhibitory affects of p21/Dacapo
Overexpression of human p21 (an inhibitor of G1-S cyclin-dependent kinases)
posterior to the MF, under the control of the GMR promoter, inhibits
S-phase entry posterior to the MF and results in a rough eye phenotype in
adults (de Nooij and Hariharan,
1995). The GMR-p21 rough eye phenotype can be modified by
reducing the dose of cell cycle regulators
(Secombe et al., 1998
),
providing a sensitive system to investigate the role of putative cell cycle
regulators. When the dosage of hfp was reduced in a GMR-p21
background, the rough adult eye phenotype was dominantly suppressed;
GMR-p21/+, hfpEP/+ eyes were larger and contained
fewer fused ommatidia (Fig. 3C)
than GMR-p21/+, +/+ eyes (Fig.
3B). Similarly, we found that the mild rough eye phenotype caused
by overexpression of the Drosophila p21/p27 homologue dacapo
(dap) was dominantly suppressed by mutation in hfp (data not
shown).
|
Given that mammalian FIR protein negatively regulates the cell cycle via Myc, and the above data showing that Hfp might normally inhibit cell cycle progression through p21/Dacapo, we tested Drosophila Myc (dMyc) for genetic interactions with p21/Dacapo. The dMyc mutant enhances the GMR-p21 phenotype (Fig. 3N females of genotype dmycP0/+;GMR-p21/+ and Fig. 3O males dmycP0/Y; GMR-p21/+ compared with the control female GMR-p21/+, in Fig. 3M). Conversely, the GMR-GAL4, UAS-dacapo reduced/rough eye phenotype is suppressed by co-expression of a UAS-dmyc transgene (Fig. 3R compared with Fig. 3P). The finding that the inhibitory affect of p21/Dacapo on the G1 to S transition can be suppressed by either reducing the dose of hfp or by overexpressing dmyc, suggests that Hfp and dMyc may have antagonistic effects on the G1 to S transition in the eye imaginal disc.
Mutation of hfp rescues the ovary phenotype and sterility of dmyc mutant females
Given that mammalian FIR protein is a negative regulator of Myc,
and the above data showing that Hfp inhibits cell cycle progression, we
investigated the possibility that Hfp regulates dmyc in
Drosophila. If the role of Hfp as a negative regulator of
dmyc has been conserved, we hypothesized that reducing the dose of
hfp might suppress the dmyc mutant phenotype. The three
characterized hypomorphic dmyc alleles,
diminutive1 (dmycdm1)
(Gallant et al., 1996),
dmycP0, and dmycP1, are all recessive
female sterile (unpublished data). Analysis of ovaries from
dmycP0 and dmycP1 females revealed
that early stage (stage 2-9) egg chambers were of normal appearance but then
arrested between stages 10-11 of oogenesis with smaller ovarioles
(Fig. 4B,D).
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DNA endoreplication in nurse cells and follicle cells also occurs during
stage 10 of oogenesis. Follicle cells undergo genomic endoreplication until
stage 10A and switch to amplification of specific loci, including the
chorion genes at stage 10B (Calvi
et al., 1998; Edgar and
Orr-Weaver, 2001
). Reduced chorion gene amplification was
observed in dmycP1/dmycP1 follicle
cells (Fig. 4J,M) compared with
the wild-type control (Fig.
4I,L). Consistent with results above, reducing the dosage of
hfp restored chorion gene amplification to normal levels in
dmycP1/dmycP1 ovaries
(Fig. 4K,N).
To test whether increased dmyc mRNA was associated with reduced
hfp gene dosage in ovaries, in-situ hybridization analysis was
performed. In wild-type egg chambers, abundant dmyc expression was
observed in nurse cells and follicle cells
(Fig. 4O,R), consistent with
previous findings (Gallant et al.,
1996). As expected, dmyc mRNA abundance was reduced in
dmycP0/dmycP0 nurse cells compared
with wild type, and was almost absent in follicle cells and the oocyte
(Fig. 4P,S). Increased
dmyc expression was observed in follicle cells from
dmycP0/dmycP0;
hfpEP/+ egg chambers compared with those from
dmycP0/dmycP0 flies
(Fig. 4Q,T). The relative
increase in dmyc mRNA in nurse cells of
dmycP0/dmycP0;
hfpEP/+ ovaries is less striking and is likely to
be a consequence of cytoplasmic dumping, which is impaired in
dmycP0/dmycP0 but occurs in
dmycP0/dmycP0;
hfpEP/+ ovaries (see above). These data suggest
that, like mammalian FIR, Hfp functions as a negative regulator of
dmyc.
Hfp mutant clones have elevated dmyc expression
To further investigate regulation of dmyc expression by Hfp, we
generated clones of homozygous hfp mutant tissue in wing imaginal
tissues using FLP/FRT-induced mitotic recombination of the
hfpEP allele (Xu and
Rubin, 1993). Analysis of hfp mutant clones revealed
reduced levels of staining with the anti-Hfp antibody in third instar eye
discs, compared with surrounding non-clonal, GFP-positive tissue (data not
shown). Previous mRNA analysis has shown that dmyc is expressed in
proliferating regions in the wing disc, with lower expression in the
non-proliferating ZNC (Johnston et al.,
1999
). Analysis of mosaic wing discs revealed elevated
dmyc mRNA expression specifically in hfpEP mutant
clones, including those spanning the ZNC, compared with surrounding
hfpEP/+ cells and wild type clones
(Fig. 4V-Z). Increased levels
of dmyc transcript were also observed in hfp mutant clones
in the eye disc (data not shown); therefore, Hfp acts to repress dmyc
transcript accumulation in Drosophila imaginal tissues.
Hfp negatively regulates stg, the rate-limiting factor for G2-M progression
The evidence above suggests that Hfp negatively regulates accumulation of
dmyc transcript; however, the finding that reducing the dose of
dmyc does not rescue the hfp hypomorphic phenotype (data not
shown), suggests that the pupal lethality associated with the hfp
mutant is not simply a consequence of increased levels of dmyc.
Therefore, if the hfp mutant lethality is not exclusively due to
increased dmyc expression, Hfp may regulate other essential
genes.
Examination of genetic interactions between dMyc and Hfp in the eye also
suggested a second role for Hfp. dmyc overexpression in wing discs
results in larger cells due to increased growth, an accelerated G1 phase and a
compensatory extension of G2 phase due to the fact that Cdc25c/Stg, the rate
limiting factor for G2-M progression, is not upregulated by dMyc
(Johnston et al., 1999).
Similarly, overexpression of dmyc using the eye driver
GMR-GAL4 results in larger cells posterior to the MF in third instar
larvae (Fig. 5G,H compared with
wild type, Fig. 5A,B), larger
adult ommatidia (Fig. 5I
compared with wild type, Fig.
5C) and an oversized adult eye
(Fig. 5J compared with wild
type, 5D). Reducing the level
of hfp in this genetic background results in a further increase in
the overall size of the dmyc overexpressing adult eye
(Fig. 5P compared with
Fig. 5J) with more
disorganized, slightly larger ommatidia
(Fig. 5M,N,O compared with
Fig. 5G,H,I).
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Indeed, as expected in the event of a G2 delay, the band of mitotic cells was reduced in GMR-GAL4, UAS-dmyc/+ eye discs (Fig. 5L compared with wild type, Fig. 5F). Reducing the dose of hfp increased both the number of S-phase cells (Fig. 5Q) and restored M-phase entry (Fig. 5R). The increased mitotic cells observed upon reducing the dose of hfp suggests that more of the dmyc overexpressing G2-delayed cells progress into mitosis. This cannot be explained by the effect of increased dmyc levels when hfp is reduced and suggests that Hfp may normally negatively regulate a cell cycle component that is required for promotion of G2-M progression.
The increased number of S-phase cells observed upon halving the dose of
hfp may be a consequence of passage of G2-delayed cells through
mitosis into another S phase. To examine the possibility that Hfp might
regulate G2-M progression via an inhibitory affect on Stg (the rate limiting
regulator of G2-M), we generated hfpEP,
stgAR2 double mutants and analysed mitoses in mutant
embryos using anti-phosphohistone H3 (PH3) staining
(Fig. 6A-H). Analysis of
hfpEP mutant embryos revealed an apparently normal pattern
of PH3 staining in cycle 16 mitotic domains when compared with wild type
(Fig. 6C compared with
6A). Closer inspection of the
mitotic figures from hfpEP mutant embryos revealed
abnormal chromosome morphology; including many lagging chromosomes that are
often mis-segregated due to closure of the contractile ring prior to sister
chromatid separation (Fig. 6D).
Maternal Stg enables mitoses prior to embryonic cycle 14; however, after
interphase 14 zygotic transcription of stg is required for G2-M
progression, and as a consequence stg mutants arrest in G2 of cycle
14 (Edgar and O'Farrell,
1990). As expected, cycle 14 stgAR2 mutant
embryos lacked PH3 staining (Fig.
6E), and were comprised solely of large G2 cells
(Fig. 6F). Strikingly, mitotic
entry was restored in hfpEP, stgAR2
double mutant embryos (Fig.
6G), and consequently cell size was restored to the wild type
range (Fig. 6H). Furthermore,
in contrast to the complete embryonic lethality of stg mutant
embryos, hfpEP, stgAR2 double mutants
survive embryogenesis and die between first and second instar. Thus, in
addition to negatively regulating dmyc and G1-S progression, these
results suggests that Hfp normally acts to negatively regulate mitotic entry
via negative regulation of stg.
|
Wg patterning regulates Hfp expression
The cell cycle arrest and repression of dmyc normally observed in
the wing disc ZNC requires Wg expression and a functional Wg pathway
(Johnston and Edgar, 1998).
Hence, expression of a dominant negative form of TCF (TCFDN) in
cells of the ZNC causes ectopic induction of dmyc and cell cycle
entry (Johnston et al., 1999
).
As both dmyc expression and cell proliferation in the wing disc
appear to be inhibited by Hfp, we hypothesized that Hfp expression may be
under the control of the Wg pathway. To test this, we activated the Wg pathway
in the posterior compartment of the wing disc by expressing a dominant
negative form of Shaggy, SggDN, using the en-GAL4 driver.
Shaggy is the Drosophila orthologue of vertebrate glycogen synthase
kinase 3 (GSK3), an inhibitory component of the Wg signalling pathway
(Siegfried et al., 1992
).
Therefore, expression of the dominant negative transgene (SggDN)
results in ectopic activation of the Wg signalling pathway. As expected,
control en-GAL4,UAS-GFP larval wing discs showed GFP expression
restricted to the posterior region of the disc and ubiquitous staining for Hfp
(Fig. 7A-C). Increased staining
with the anti-Hfp antibody was observed in the posterior region of
en-GAL4,UAS-hfp wing discs as expected
(Fig. 7D-F). Significantly,
activation of the Wg pathway by SggDN resulted in similar high
levels of ectopic Hfp expression in the posterior region of the wing disc
(Fig. 7G-I). To further confirm
that Hfp is upregulated by Wg signalling, we analysed axin mutant
clones, in which the Wg pathway is constitutively active, since Axin normally
downregulates Armadillo (Hamada et al.,
1999
). Indeed, increased Hfp protein was observed in
d-axin mutant clones, marked by the absence of GFP
(Fig. 7J-L). Conversely, when
Wg signalling was blocked by expressing a dominant negative form of TCF
(TCFDN) in the ZNC using the C96-GAL4 driver
(Johnston et al., 1999
) Hfp
protein was reduced in all TCFDN expressing cells, which are marked
by coexpression of GFP (Fig.
7N,O, compared with the normal high level of Hfp in ZNC cells from
wild type, Fig. 7M). Reduced
numbers of ZNC cells (i.e. fewer cells stained for GFP) are observed as a
consequence of TCFDN overexpression, since cells die by apoptosis
when Wg signalling is blocked (Johnston
and Sanders, 2003
). Therefore, ectopic activation of the Wg
pathway is associated with increased levels of Hfp in the wing disc, and
blocking Wg signalling reduces Hfp expression. Taken together, these results
show that ectopic activation of the Wg pathway increases the level of Hfp in
third instar wing discs, consistent with the notion that Wg may normally act
by inducing hfp to inhibit dmyc expression in the ZNC.
|
![]() |
Discussion |
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Dual function for Hfp in regulation of splicing and dmyc transcription?
Increased levels of dmyc transcript were observed in hfp
mutant clones, consistent with Hfp acting to repress dmyc transcript
accumulation in Drosophila imaginal tissues. The upregulation of
dmyc mRNA in hfp mutant tissue could occur through
alterations in dmyc transcription (initiation or elongation),
pre-mRNA splicing, mRNA message stability or a combination of these processes.
Mammalian FIR was first shown to regulate pre-mRNA splicing by binding to RNA
polypyrimidine tracts and cooperating with the essential splicing factor U2AF
(Page-McCaw et al., 1999).
Consistent with this, recent studies in Drosophila show that the FIR
orthologue Hfp is required for correct splicing of several genes in the
developing ovary (Van Buskirk and
Schupbach, 2002
). Mammalian FIR has been shown to have a second
role as transcriptional repressor of Myc, through first forming a
complex with the Myc activator FBP and interfering with the basal
transcription apparatus by then binding TFIIH, thereby disrupting helicase
function (Liu et al., 2000
).
The data described here suggest that the cell cycle inhibitory function of Hfp
is partly a consequence of negatively regulating dmyc expression.
Therefore, the dual roles of transcription regulation and mRNA splicing appear
to have been evolutionarily conserved between Drosophila Hfp and
mammalian FIR. It remains to be determined whether Hfp inhibits dmyc
expression by a mechanism analogous to the mammalian FIR/FBP/FUSE interaction.
A FUSE element has not been identified upstream of the dmyc promoter,
and although the Drosophila splicing factor PSI is a highly conserved
orthologue of FBP (Labourier et al.,
2002
), it has not been reported whether PSI can activate
dmyc expression.
Hfp regulates G2-M progression, via negative regulation of stg
Our finding that hfp mutants do not phenocopy dmyc
overexpression suggested that inhibition of dmyc expression is not
the only role of Hfp. Although increased S phases are observed in hfp
mutant wing discs, this is not associated with increased cell size, as occurs
with dmyc overexpression in the wing disc. Rather, in hfp
mutant wing discs the ZNC, which normally contains domains of G1- and
G2-arrested cells (Johnston and Edgar,
1998), has ectopic S-phase and M-phase cells. Since cells in
hfp mutant wing discs are of normal size and ectopically enter S
phase, it is possible that progression through G2 may also be accelerated.
Indeed, the increased number of mitotic cells observed in eye imaginal discs
when the level of Hfp is reduced in a dmyc overexpression background,
suggests that Hfp normally negatively regulates G2-M phase progression.
Furthermore, the abnormal mitotic figures observed in
hfpEP mutant embryos are consistent with accelerated cell
cycle progression (Quinn et al.,
2001
). Most importantly, the hfp mutant rescued the cycle
14 G2-arrest that normally occurs in stg mutant embryos, and
hfp mutant clones have increased levels of Stg protein, suggesting
that Hfp normally exerts an inhibitory affect on G2-M progression via
negatively regulating Stg translation or protein stability. Thus, Hfp may be
required for negatively regulating both the G1-S phase transition by
downregulating dmyc and the G2-M transition by negatively regulating
stg.
Regulation of Hfp, dMyc and Stg by the Wingless pathway
The Wg pathway is required to downregulate both dmyc and
stg expression in order to limit cell proliferation in the ZNC during
wing development (Johnston and Edgar,
1998; Johnston et al.,
1999
). Activation of the Wg pathway, using either dominant
negative Shaggy or by generation of axin clones, resulted in a strong
and specific increase in Hfp protein, demonstrating that Wg pathway activation
is sufficient to cause Hfp induction. Our findings supported a model in which
Wg signalling causes induction of Hfp in the wing disc ZNC, which in turn
inhibits dmyc expression (to elicit the posterior, G1 arrest) and
stg expression or activity (to provide the anterior, G2-arrested
domains) (Fig. 8). The
involvement of Achaete and Scute in this process, which have been previously
shown to play a role in the negative regulation of stg
(Johnston and Edgar, 1998
)
remains to be elucidated. Previous studies have shown that Ras signalling
through Raf/MAPK upregulates dmyc post-transcriptionally in wing disc
cells and is required to maintain normal dMyc protein levels in the wing disc
(Prober and Edgar, 2000
;
Prober and Edgar, 2002
). In
contrast, since hfp clones have increased dmyc mRNA, Hfp
must normally inhibit dmyc mRNA accumulation. Furthermore,
overexpression of Hfp inhibits cell proliferation in all wing and eye imaginal
discs, suggesting that Hfp may normally override mitogenic signals and lead to
cell cycle arrest during particular stages of development.
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ACKNOWLEDGMENTS |
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