Department of Biochemistry, University of Washington, J591, HSB, Seattle, WA 98195-7350, USA
Author for correspondence (e-mail:
hannele{at}u.washington.edu)
Accepted 16 March 2004
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SUMMARY |
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Key words: Drosophila, Notch, String, Fizzy related, Dacapo, Cell cycle, Follicle cells
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Introduction |
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In normal mitotic cells cyclin-dependent kinases (Cdks) induce
phosphorylation events that control whether the cell enters the M- or S-phase.
CycA/Cdk1 and CycB/Cdk1 complexes that can be activated by Cdc25-type
phosphatases mediate M-phase control. S-phase is controlled by other
complexes: CycE/Cdk2, CycA/Cdk2, and CycD/Cdk4 or Cdk6. Some of these
complexes are known to be negatively regulated by several Cdk inhibitors, such
as p27Kip1. p27Kip1 inhibits CycE/Cdk2 complexes and thereby arrests cells in
the G1-phase (Olashaw and Pledger,
2002).
Cells normally progress through the cell cycle in a series of sequential
steps in which each step is dependent on the proper completion of the previous
stage. However, endocycling cells are exceptions to this rule because they
proceed to S-phase without completing M-phase. How this occurs is not
understood. In some endocycling cells, bypassing the mitotic stage correlates
with eliminating or reducing the Cdk activity required for M-phase entry. For
example in Drosophila early embryonic cells and follicle cells,
components and regulators of the M-phase can be eliminated at a
transcriptional level and/or at a posttranscriptional level
(Deng et al., 2001;
Sauer et al., 1995
;
Sigrist and Lehner, 1997
).
Posttranscriptional modulations are often mediated by ubiquitination via the
APC-complex with degradation of the cyclins and securins
(Peters, 2002
;
Vodermaier, 2001
).
The key question in endocycle regulation is how the transition from the
mitotic phase to the endocycle is controlled. Two signaling pathways have been
identified as regulators of the mitotic-to-endocycle transition: the
thrombopoetin pathway, which acts during differentiation of megakaryocytes and
the Notch pathway, which acts during Drosophila oogenesis and during
the differentiation of trophoblasts (Deng
et al., 2001; Lopez-Schier and
St Johnston, 2001
; Nakayama et
al., 1997
; Wu et al.,
2003
; Zimmet and Ravid,
2000
). Human teratocarcinomas also seem to arise from defects in
the mitotic-to-endocycle transition in trophoblasts
(Cross, 2000
). The key
cell-cycle targets of these pathways, however, remain elusive.
In Drosophila follicle cells the function of the Notch pathway in
the mitotic-to-endocycle transition has been well established
(Deng et al., 2001;
Lopez-Schier and St Johnston,
2001
). Specifically, the ligand Delta is secreted by germ line
cells and activates Notch in the follicle cells. Subsequently, the cytoplasmic
portion of Notch is cleaved by Presenillin and moves to the nucleus where, in
combination with a transcription factor, Suppressor of Hairless [Su(H)], it
affects the transcription of various target genes. Lack of Notch activity in
Drosophila follicle cells leads to prolonged mitosis at the expense
of endocycles. This has led to the suggestion that Notch functions in this
context as a tumor suppressor (Deng et
al., 2001
; Lopez-Schier and St
Johnston, 2001
). Because very few signaling pathways have been
identified that stop the mitotic cell cycle, it is important to understand in
detail the relationship between the Notch pathway and known cell cycle
regulators.
We have previously shown that one of the cell cycle components that
responds to Notch activity at the transcriptional level, String/Cdc25
phosphatase, which is a regulator of the transition between S- and M-phase, is
not sufficient, by itself, to keep all the cells in mitotic phase, suggesting
that other components are needed for the mitotic-to-endocycle transition
(Deng et al., 2001;
Schaeffer et al., 2004
). One
such Notch-regulated component, essential for the mitotic-to-endocycle
transition is Hec1/CdhFzr, a WD40-domain regulator of the
APC-Ubiquitination complex. Hec1/CdhFzr, which acts with other
cellular components important for M-phase entry, is dispensable for mitosis
but essential for the mitotic-to-endocycle transition in follicle cells
(Schaeffer et al., 2004
).
However, cells in fzr/ clones do not prolong mitosis
unless accompanied with ectopic string. We have now shown that this
APC regulator is sufficient to induce, albeit with low penetrance, premature
endocycles if precociously expressed in the follicle cell epithelium. We
furthermore identify an inhibitor of the CyclinE/CDK complex, Dacapo, that is
reduced because of Notch activity in the mitotic-to-endocycle transition as a
repressor of endocycles in follicle cells. Notch activity, therefore, executes
the mitotic-to-endocycle transition by regulating three cell cycle
transitions: repression of String blocks M-phase, activation of
Hec1/CdhFzr allows G1 progression, and repression of Dacapo assures
entry into S-phase.
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Materials and methods |
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Generation of follicle cell clones
Drosophila melanogaster stocks were raised on standard
cornmeal-yeast-agar-medium at 25°C. To obtain follicle cell clones,
1-to-5-day-old flies were heat-shocked as adults for 50-60 minutes at 37°C
and put in freshly yeasted vials for 3 or 5 days. To obtain germ line clones,
flies were heat-shocked as second and third instar larvae for 2 hours on 2
consecutive days. Once they emerged as adults, they were placed in vials with
fresh yeast paste for 1-5 days prior to dissection.
Nuclear preparation and flow cytometric analysis
Nuclear preparation was done essentially as described in Bosco et al.
(Bosco et al., 2001) and Calvi
and Lilly (Calvi and Lilly,
2004
) with minor modifications. Ovaries from 50-100 females were
incubated in 1 ml of 5 mg/ml collagenase (Blend type H, Sigma-Aldrich C8051)
in 90% Graces insect medium at 4°C for 15 minutes on a Clay-Adams Nutator.
Ovaries were then disrupted by pipeting them several times through a P-1000
tip and then pelleted in an Eppendorf centrifuge by a 2-second spin at 1000
g. The pellet was resuspended in 500 µl of buffer A (15 mM
Tris-HCl, pH 7.4, 60 mM KCl, 15 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.15 mM
spermine, 0.5 mM spermidine) plus 0.25 M sucrose and 0.5% NP-40. Next the
ovaries were homogenized at 4°C by 10 strokes in a 2 ml Kimble/Kontes
glass dounce with a glass B-clearance pestle. The resulting lysate was cleared
by serial passage through 150-, 100-, 50- and finally 30-µm Nitex filters
(Sefar America, Depew, NY, USA). Nuclei were then pelleted by passage through
a sucrose (0.25 M-2.5 M, in 1X Buffer A) density step gradient 15,000
g for 20 minutes at 22°C in a Beckman TLS-55 rotor and
resuspended in 200 µl of buffer A that contained 0.1% NP-40 and 20 µg/ml
propidium iodide. Follicle cells nuclear ploidy was determined by
fluorescence-activated cell sorting (FACS) analysis using a Becton Dickinson
FACScan cytometer by numbering the intensity of propidium fluorescence in
stained nuclei. Nuclei were exited with a 488 nm laser, and the emission was
monitored through a 585/42 nm band pass filter. Results were analyzed by using
CellQuest and Multicycle software.
Staining procedures
Ovaries were dissected in phosphate-buffered saline (PBS) and fixed while
shaking on a nutator for 10 minutes in PBS containing 5% Formaldehyde. Next,
they were rinsed with PBT (PBS/0.2% Triton X-100) four times (15 minutes, each
rinse) and blocked in PBTB (PBT, 0.2% BSA, 5% Normal Goat Serum) for one hour
at room temperature. The tissue was incubated with primary antibodies
overnight at 4°C. The next day they were rinsed with PBT four times (15
minutes, each rinse) and blocked in PBTB for one hour at room temperature. The
ovaries were then incubated in secondary antibodies overnight at 4°C. The
next day they were rinsed with PBT (4x15 minutes) and stained with DAPI
(1 µg/ml in PBT) for 10 minutes. Finally, they were washed with PBT twice
four times (5 minutes, each wash) and dissected onto slides in 70% glycerol,
2% NPG, 1X PBS.
Follicle cells were labeled with BrdU as described previously
(Bosco et al., 2001;
Calvi and Lilly, 2004
;
Lilly and Spradling, 1996
)
with slight modifications. Ovaries were dissected in Graces insect
medium and then incubated with 10 µM BrdU (Boehringer Mannheim) in the same
medium for 1.5 hours at room temperature. The ovaries were then fixed in 10.5%
formaldehyde for 15 minutes, washed with PBT, then treated with 2N HCl for 45
minutes. Sodium borate (100 mM) was used for neutralization. The tissues were
then rinsed with PBT three times (10 minutes, each rinse) and blocked in PBTB
for half an hour at room temperature and incubated with mouse anti-BrdU
antibodies (Becton Dickinson) overnight at 4°C. The next day, ovaries were
rinsed with PBT six times (5-10 minutes, each rinse), blocked in PBTB for 30
minutes at room temperature and incubated with secondary antibodies for 2
hours at room temperature. Thereafter, the ovaries were rinsed with PBT four
times (15 minutes, each rinse) and stained with DAPI for 10 minutes. Finally,
they were washed with PBT twice (5 minutes, each wash) and dissected onto
slides in 70% glycerol, 2% NPG, 1X PBS.
Confocal microscopy, X-gal staining and in situ hybridization were
performed as described previously (Keller
Larkin et al., 1999; Tworoger
et al., 1999
). For in situ RNA hybridization studies stg
cDNA (LD47579) was labeled with fluorescine, whereas cycD (LD22957),
cycE (LD22682), cycA (LD44443), cycB (LD23613), and
fzr (LD21270) cDNAs were labeled with digoxigenin (all cDNAs were
from the Berkeley Drosophila UniGene Collection). A two-photon laser-scanning
confocal microscope (Leica TCS SP/MP) was used in this study.
The following primary antibodies were used at the designated dilutions: mouse anti-Fasciclin III (1:20), mouse anti-CycA (1:20) and mouse anti-CycB (1:20) from Developmental Studies Hybridoma Bank, rabbit anti-CycA [1:100, David Glover (this antibody showed CycA downregulation at stage 6 but some immunoreactive material was later observed at stage 8)], mouse anti-CycE (1:5, a gift from Helena Richardson), guinea pig anti-CycE (1:500, a gift from Terry Orr-Weaver), rabbit anti-PH3 (1:200, Upstate Biotechnology), rabbit anti-Fizzy-related (1:800, a gift from Christian Lehner), mouse or rabbit anti-ß-gal (1:5000, Sigma) and mouse anti-c-Myc (1:50, Calbiochem). The following secondary antibodies were used at the designated dilutions: Alexa 488, 568 or 633 goat anti-mouse (1:500), Alexa 488, 568 or 633 goat anti-rabbit (1:500, Molecular Probes), Alexa 488 goat anti-guinea pig (1:500, Molecular Probes).
Studying the Cyclin D role in mitotic-to-endocycle transition
We analyzed the expression pattern of CycD during mitotic-to-endocycle
transition, because it has been shown to be critical for cell growth in
Drosophila (Chen et al.,
2003; Datar et al.,
2000
; Meyer et al.,
2000
) and the cell growth is a major component of endocycle.
Surprisingly, a clear downregulation of cycD RNA level was observed
at the onset of endocycles, stage 6 (data not shown). The functional relevance
of this downregulation is not clear because overexpression of the protein, in
combination with its kinase Cdk4, does not dramatically affect entry into
endocycles. During these overexpression studies, we observed no difference in
the size of nuclei compared with those in wild-type cells and there was no
upregulation of CycB or PH3 (data not shown).
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Results |
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In the Drosophila ovary both the germ line and somatic cells arise
from stem cell populations located in an anterior ovary structure called the
germarium (Fig. 1A). At the
posterior end of the germarium, somatic cells encapsulate a 16-cell cyst of
germ line cells, a configuration in which the oocyte will eventually develop
during a three-day period (this developmental process has been divided into 14
stages). Meanwhile, the somatic follicle cells will undergo three tightly
developmentally controlled cell cycle modifications
(Calvi et al., 1998). First,
these epithelial cells undergo a mitotic division program that gives rise to
approximately 1000 follicle cells by stage 7 in oogenesis
(Fig. 1A, part I). At this
mid-oogenesis point, signaling through the Notch pathway stops the mitotic
cycles in the follicle cells and allows them to enter endoreplication to
become polyploid (Deng et al.,
2001
; Lopez-Schier and St
Johnston, 2001
) (Fig.
1A, part II). After stage 6, the follicle cells then undergo three
endocycles to become polyploid. Later in oogenesis at stage 10B in response to
unknown developmental signals, four different loci, encoding several different
genes two of which are chorion genes, synchronously initiate a gene
amplification event, that increase their copy number
(Fig. 1A, part III). During
this phase all other genomic replication origins remain inactive
(Orr-Weaver, 1991
;
Spradling, 1999
). The chorion
genes encode the eggshell proteins and amplification of these genes is needed
to produce sufficient chorion protein for a normal eggshell. These three
replication patterns are readily observed by BrdU analysis
(Calvi et al., 1998
)
(Fig. 1B). In addition, these
cell cycle programs can be distinguished by different markers, for example,
CycB and PH3 (Deng et al.,
2001
) (Fig.
1C).
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Ectopic Hec1/CdhFzr affects mitotic-to-endocycle but not endocycle-to-amplification transition
After three rounds of endocycles, the follicle cells synchronously initiate
a chorion gene amplification event that continues to increase the copy number
of four different loci. The amplification occurs by the initiation of repeated
rounds of DNA replication and fork movement to produce a gradient of amplified
DNA extending 100 kb (Calvi and
Spradling, 1999
; Orr-Weaver,
1991
). How the onset of the endocycle-to-amplification transition
is regulated is not understood.
To further analyze whether the larger nuclei observed upon ectopic fzr expression was because of precocious endocycling during the mitotic phase and/or prolonged endocycling during the amplification phase, we analyzed whether the amplification stage was defective or delayed in cells that overexpressed fzr. BrdU incorporation revealed normal amplification patterns in control and fzr-overexpressing cells (Fig. 2F), suggesting that the extra endoreplication observed upon fzr overexpression was not caused by defects in the endocycle-to-amplification transition. We therefore conclude that the larger nuclei arose because of a premature switch in the timing of the mitotic-to-endocycle transition.
Myc in the mitotic-to-endocycle transition
Premature expression of Fzr is sufficient to halt mitotic cell cycles 60%
of the time, however, only one-third of these cells will enter premature
endocycles (Fig. 2B,C). One
possible explanation for these results is that components at G1/S transition,
such as G1 cyclins, further restrict from entering the premature endocycles by
blocking entry into the S-phase in mitotic cells.
In other systems G1/S transition is controlled in part by factors that
regulate cell growth such as CycD or Myc
(Frei and Edgar, 2004).
Curiously, CycD does not seem to be critical for endocycles in follicle cells
(see Materials and methods). This presents a paradox because CycD is known to
be critical for cellular growth in Drosophila
(Datar et al., 2000
;
Meyer et al., 2000
) and a key
feature of all endocycles is an increase in cell size. Interestingly, the
expression screen described previously
(Table 1) (Bourbon et al., 2002
) might
provide an answer to this apparent paradox, because this screen demonstrated
that Myc, another component required for growth, is transcriptionally
upregulated at the transition.
dMyc is the Drosophila homologue of the BHLHZ Myc-oncogene family
of transcription factors. Recent studies have shown that dMyc has a function
in cellular growth (Iritani and Eisenman,
1999; Johnston and Gallant,
2002
; Johnston et al.,
1999
). To test whether dMyc can influence the mitotic-to-endocycle
transition, we overexpressed dmyc in developing follicle cells and
analyzed cell cycle markers in both cell cycle programs. No obvious change was
observed during the mitotic stage upon ectopic expression of dmyc
(Fig. 2C); however during the
endocycling stage, cells overexpressing dmyc were larger than the
neighboring wild-type cells (Fig.
2E). In particular, larger nuclei were observed in 32% and 55% of
the cells at stage 7-9 and 12 (respectively) after overexpressing
dmyc (Fig. 2B). FACS
analysis revealed that these large nuclei were indicative of extra endocycles
because cells with 32n ploidy were observed among these follicle cells
(Fig. 2K). Similar to the case
of fzr overexpression, BrdU incorporation revealed normal
amplification patterns during stage 12 in oogenesis in cells overexpressing
myc (Fig. 2G),
suggesting that the extra endoreplication observed during myc
overexpression was not caused by defects in the endocycle-to-amplification
transition. Because the higher ploidy accompanies no change in the timing of
mitotic-to-endocycle or endocycle-to-amplification transition, we conclude
that this defect was caused by faster endocycle kinetics than in wild-type
follicle cells. Thus, these data suggest that Myc-dependent growth can
regulate endocycle kinetics in follicle epithelial cells.
Proper CycE regulation is required for the mitotic-to-endocycle transition
Another G1/S-regulator that might play a role in the proper
mitotic-to-endocycle transition in follicle cells is Cyclin E
(Calvi et al., 1998).
Drosophila Cyclin E forms a complex with the DmCdc2c/Cdk2 kinase and
controls the progression through the S phase; its downregulation limits
embryonic proliferation and its oscillation is required for endocycling
(Follette et al., 1998
;
Knoblich et al., 1994
;
Weiss et al., 1998
). The
cycE mRNA expression pattern in the follicle cells during oogenesis
reflects a continuous requirement both in mitotic and endocycles without
downregulation at the mitotic-to-endocycle transition; but rather, an equal
level of patchy cycE mRNA levels within the follicle
cells throughout oogenesis (Fig.
3A). The same patchy pattern of CycE protein level
is seen in mitotic and endocycling follicle cells, indicating the cell
cycle-dependent oscillation of CycE protein. However, we observed a clear, but
not complete, downregulation of CycE protein levels at approximately stage 7,
just after the follicle cells stopped expressing CycB and PH3 (mitotic
markers) (Fig. 3B,C). In
follicle cell clones mutant for a transcription factor in the Notch pathway,
Su(H), the CycE levels were somewhat abnormally regulated during endocycles,
which suggested a role for the Notch signaling pathway in this
posttranscriptional regulation (data not shown).
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|
Dacapo downregulation by Notch signaling at mitotic-to-endocycle transition is required for proper endocycling
The Cip/Kip families of cyclin-dependent kinase inhibitors (CKI) are the
main effectors linking developmental programs and cell cycle progression
(Liu et al., 2002).
Drosophila dacapo (dap) encodes an inhibitor of cyclin
E/cdk2 complexes with similarity to the vertebrate Cip/Kip inhibitors
(Lane et al., 1996
;
Liu et al., 2002
;
Meyer et al., 2002
) and
thereby inhibits entry into the S-phase. In accordance with this role,
mammalian family members are upregulated during checkpoint activation and are
expressed in terminally differentiated tissues. Dacapo contains a conserved
amino-terminal domain that binds to and inhibits all kinases involved in G1/S
transition. This inhibition is achieved by a conformational change in the
cyclin/Cdk complex (Pavletich,
1999
).
In follicle cells, the in situ mRNA hybridization pattern for
dacapo indicates that its transcriptional level is downregulated at
stage 6-7 (Fig. 4A),
suggesting a potential regulation by the Notch pathway. Because
dacapo has previously been shown to be developmentally controlled by
an extensive promoter region, we used different dap-constructs
(Liu et al., 2002;
Meyer et al., 2002
) and
studied their expression patterns in the follicle cells during oogenesis. The
smallest constructs, that faithfully represent dacapo mRNA pattern
(dap5gm and dap6gm), include the entire gene as well as 1.8 kb and 2 kb of the
promoter region fused with a myc-epitope tag. These transgenic lines
showed clear myc-epitope staining in the follicle cells before stage 6 and a
downregulation of expression thereafter
(Fig. 4B). One of these
constructs, dap5gm, was used as a marker for Dacapo expression in the
following studies.
|
To analyze whether the Notch-dependent repression of dacapo was
important for the mitotic-to-endocycle transition, we tested the functional
consequence of dacapo loss-of-function and prolonged expression for
follicle cell cycle control. Dacapo loss-of-function clones revealed
no obvious phenotype; nuclei sizes were normal and endocycle did not appear to
be inhibited. Overexpression of dacapo has previously been shown to
inhibit entry into late amplification
(Calvi et al., 1998) and, in
the salivary gland, overexpression of dacapo inhibits endoreplication
(Weiss et al., 1998
). We
likewise observed cell cycle defects because of the prolonged expression of
dap: smaller nuclei and a failure to incorporate BrdU, indicating a
lack of S-phases (Fig. 4D-F).
CycA and CycB, although upregulated in cells overexpressing CycE past stage 6,
were not upregulated in cells overexpressing dap
(Table 2). These results
suggest that Notch-based downregulation of dacapo is critical for
endocycling because overexpression of dacapo, a CIP/KIP-type
cyclin-dependent kinase inhibitor of Cdk2/CycE complexes, can halt the
follicle cell endocycles at the apparent G1/S transition. Also, one
possibility is that Dacapo in mitotic cells aborts premature attempts to enter
endocycles and precocious expression of Hec1/CdhFzr
(Fig. 2A,C).
Ago is essential for endocycles but dispensable for mitotic cycles
Because dacapo is downregulated after the mitotic-to-endocycle
transition, we investigated whether archipelago, a second regulator
of CycE protein level was required for proper oscillations of CycE during
endocycles. Archipelago is an F-box protein with seven tandem WD repeats that
recognizes auto-phosphorylated CycE. Ago protein binds directly to Cyclin E
and targets it for ubiquitin-mediated SCF-dependent degradation
(Moberg et al., 2001;
Strohmaier et al., 2001
). The
in situ hybridization pattern of archipelago reveals continuous mRNA
expression both in the mitotic and endocycling stages
(Fig. 5A). To investigate the
role of Archipelago in these cells we made follicle cell clones with three
different ago alleles (Moberg et
al., 2001
). As expected, we saw an increase in CycE protein in
most mutant follicle cells at all stages during oogenesis in all three mutants
(Fig. 5B). We then analyzed the
effect these mutations had on both mitotic and endocycling cells. Based on
phenotypic analysis in the Drosophila eye, we might expect
overproliferation of cells mutant for ago
(Moberg et al., 2001
). Yet, we
observed no obvious defects in the control of mitotic divisions in
ago clones: the ratio between the number of cells in sister clone
versus mutant clone was 1:0.9 (n=14) and the BrdU incorporation and
CycA and CycB levels during mitotic divisions were normal
(Fig. 5D and data not shown).
In addition, the expression patterns of PH3 and String in ago clones
were indicative of a normal halt in mitotic cycles at stage 6
(Fig. 5C).
|
Notch controls independently String, Hec1/CdhFzr and Dacapo expression
Notch activity affects the expression of Stg
(Deng et al., 2001), Fzr
(Schaeffer et al., 2004
) and
Dacapo (this study) at the mitotic-to-endocycle transition in follicle cells.
Because cell cycle regulators can control each other
(Futcher, 2002
), we tested
whether changes in these three Notch-dependent cell cycle regulators lead to
changes in each others expression levels. In this scenario one of the
targets might be the primary responder, whereas the others would be downstream
components of the pathway.
In particular, because Hec1/CdhFzr loss-of-function phenotype is
similar to the phenotype observed upon altering Cyclin E activity
(Schaeffer et al., 2004) (Figs
3,
4,
5), we tested whether defects
in Cyclin E levels can result in downregulation of Hec1/CdhFzr.
Overactivation of Cyclin E (because of lack of ago function or
overproduction of CycE) blocks the endocycling cells at G2, whereas
inactivation of Cyclin E (because of overproduction of Dacapo) blocks them in
G1 (Table 2). However,
importantly, neither of these alterations result in repressed Fzr expression
at the mitotic-to-endocycle transition: cells that do not endocycle because of
overexpression of dacapo or cyclin E show normal
Hec1/CdhFzr levels at stages 7-9
(Fig. 6A,B). Based on these
data, block of endocycles by CycE or Dacapo overexpression does not repress
the expression of the endocycle regulator, Hec1/CdhFzr. However
accumulation of CycA and CycB are observed upon Cyclin E overexpression
(Table 2, Fig. 3D,E), suggesting that
even though Hec1/CdhFzr is present it might not be in an active
form.
|
Based on these findings, no obvious feedback regulation was observed between these cell cycle regulators, disturbed CycE levels did not block endocycles by repressing Hec1/CdhFzr expression levels and premature Fzr does not block mitosis by affecting String levels. Therefore, these data suggest that the Notch-dependent regulation of these targets is independent of each other (Fig. 7A).
|
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Discussion |
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Hec1/CdhFzr
The exit from mitosis and/or progression through G1 requires the
inactivation of cyclin-dependent kinases, mediated by the APC/C-dependent
destruction of cyclins (Sigrist et al.,
1995; Sorensen et al.,
2001
). APC/C is regulated by multiple mechanisms, such as
phosphorylation and by spindle checkpoints. Key factors for APC/C function and
regulation are the WD proteins Cdc20 and Hec1/Cdh. These proteins seem to bind
directly to substrates and recruit them to the APC/C core complex.
Importantly, Cdc20 and Hec1/Cdh bind and activate APC/C in a sequential manner
during mitosis. APC/C-Cdc20 is activated at the metaphase/anaphase transition,
and gets replaced by APC/C-Hec1/Cdh in telophase. This second complex remains
active in the subsequent G1 phase.
In Drosophila the homologue of Hec1/Cdh, Fzr, also induces the
APC/C-complex-dependent proteolysis of CycA and B and is required for the
G1-phase progression (Jacobs et al.,
2002; Sigrist and Lehner,
1997
). Fzr is required for cyclin removal during G1 when the
embryonic epidermal cell or follicle epithelial proliferation stops and the
cells enter endocycles (Schaeffer et al.,
2004
; Sigrist and Lehner,
1997
).
We now show that premature Hec1/CdhFzr transcription in follicle cells is sufficient to block mitosis and initiate precocious endocycling. This suggests that Fzr is a powerful player in the mitotic-to-endocycle switch, yet regulation of other components is also required for the efficiency of this process. Regulators of G1-S transition, such as Dacapo/CIP/KIP, which also turns out to be a Notch-regulated component, possibly abort premature attempts by follicle cells to enter the endocycle.
CycD or Myc in growth control
Our data suggest that a component regulating growth and thereby the
kinetics of G1/S transition in follicle cell endocycles is the Myc oncogene
instead and independent of CycD. In mammals c-Myc controls the decision to
divide or not to divide and thereby functions as a crucial mediator of signals
that determine organ and body size
(Levens, 2003;
Trumpp et al., 2001
).
Interestingly, overexpression of dmyc in follicle cells does not
affect the mitotic cycles but induces, instead, extra endocycles. Because the
timing for entering and exit from the endocycles has not changed, however,
increased ploidy is observed, we suggest that the rate of endocycles is
increased because of the overexpression of Myc. This finding is in accordance
with recent loss-of-function analysis on myc in follicle cells,
suggesting that myc mutant follicle cells can make the transition
from mitosis to the endocycle, but that they can only very inefficiently
support the endocycle (Maines et al.,
2004
). Therefore, both loss-of-function and overexpression
experiments suggest that Myc is an essential component for the proper rate of
endocycles in follicle cells.
Cyclin E in endocycles
In addition to Myc and Cyclin D, Cyclin E also plays an important role in
the regulation of the G1/S-transition. Cyclin E binds to and activates the
cyclin-dependent kinase Cdk2, and thereby promotes the transition from G1 to S
(Knoblich et al., 1994).
Oscillation of Cyclin E activity is a mechanism responsible for the timely
inactivation of this G1 cyclin/Cdk complex and an arrest in cell
proliferation. The oscillation of Cyclin E level is controlled partly by a
SCF-ubiquitin-dependent proteolysis (Koepp
et al., 2001
; Moberg et al.,
2001
; Strohmaier et al.,
2001
; Won and Reed,
1996
). Fluctuations of Cyclin E are critical for multiple rounds
of endocycles (Follette et al.,
1998
; Weiss et al.,
1998
).
Cyclin E is critical for endocycles in follicle cells as well, and our
analysis shows that the CycE level is controlled by an SCF-regulator, F-box
protein, Ago/hCdc4/Fbw7. Fbw7 (Ago) associates specifically with
phosphorylated Cyclin E, and catalyzes Cyclin E ubiquitination in vitro
(Koepp et al., 2001).
Depletion of Ago leads to accumulation and stabilization of Cyclin E in vivo
in human and D. melanogaster. This leads to increased mitosis in
certain mammalian and Drosophila cell types. In addition,
ago loss-of-function clones in the germ line will cause extra mitotic
divisions or, in contrast, cell cycle arrest and polyploidy
(Doronkin et al., 2003
).
However, we have shown that increased Cyclin E levels observed in ago
loss-of-function mutant clones do not affect the mitotic cycles in follicle
cells but do halt the transition to endocycles that normally occurs at stage
6.
Why is the function of Ago/hCdc4/Fbw7 critical to endocycles but not to mitotic cycles in follicle epithelial cells? A potential answer might reside in Dacapo, a CIP/KIP-type inhibitor of Cyclin E/Cdk2 complexes that is regulated in the mitotic to endocycle transition by activation of Notch pathway. We have shown that dacapo is downregulated at mitotic-to-endocycle transition because of Notch activation and ectopic expression of dacapo represses endocycle progression. It is plausible that during mitotic phases Ago and Dacapo share a redundant role for regulating the Cyclin E activity level, however, dacapo is downregulated by Notch pathway at the time of mitotic-to-endocycle transition and at that point Ago gains the critical role of sole regulator of Cyclin E protein activity level. However, downregulation of Dacapo does not readily explain the reduction of CycE levels observed in mitotic-to-endocycle transition (Fig. 3C). We detected elevation of CycE protein level in response to Dacapo overexpression, pointing out that this CKI may stabilize CycE in an inactive form. One possibility therefore is that less CycE protein is observed after the Dacapo downregulation because Dacapo is no longer stabilizing it.
Why is Dacapo downregulated at the time of endocycle transition? Expression
of Dacapo is important for proper cell cycle regulation. For example, during
vertebrate development, members of the CIP/KIP family of CKIs are often
upregulated as cells exit the mitotic cycle and begin to terminally
differentiate. Also, reduced expression of p27Kip1 was frequently shown to
correlate with a poor prognosis in various cancers
(Fredersdorf et al., 1997;
Geisen et al., 2003
), and in
the absence of p21, DNA-damaged cells arrest in a G2-like state, but then
undergo additional S-phases without intervening normal mitoses. They thereby
acquire grossly deformed, polyploid nuclei and subsequently die through
apoptosis (Waldman et al.,
1996
). Also, p21 elimination causes centriole overduplication and
polyploidy in human hematopoietic cells
(Mantel et al., 1999
). In the
Drosophila germ line Dap is differentially regulated in the nurse
cells versus the oocyte. High Dap levels in the oocyte are critical to the
maintenance of the prophase I meiotic arrest and ultimately to later events of
oocyte differentiation, and in the nurse cells the oscillations of Dap drive
the endocycle (Hong et al.,
2003
). In contrast to all these examples, in endocycling follicle
cells reduction of p21/Dacapo is a requirement for normal endocycle
progression. Similarly, in a megakaryocytic cell line, differentiation is
correlated with a downregulation of p27
(Fredersdorf et al., 1997
). We
propose that the downregulation of Dacapo is a reasonable strategy to bypass
the G1/S transition and to enter endocycling when mitosis is not completed,
however, how these endocycling cells escape possible centrosome amplification
and apoptosis that could be consequences of the lack of Dacapo/p21-activity is
not clear. This diversity in the processes, that allow cells to exit from
mitotic cell cycle, is generating or representing regulatory multiplicity that
might be reflected in the ways eukaryotic cells acquire tumor formation
capacity.
Notch as a tumor suppressor
Recent findings by Rangarajan et al. and Nicolas et al.,
(Nicolas et al., 2003;
Rangarajan et al., 2001
) have
shown that Notch acts as a tumor suppressor in mouse skin epithelium. Ablation
of Notch results in epidermal and corneal hyperplasia followed by the
development of skin tumors and facilitated chemical-induced skin
carcinogenesis. In these cell types Notch1 deficiency results in increased and
sustained expression of Gli2 and derepression of beta-catenin. Therefore the
authors have suggested, that in mouse skin epithelium Notch pathway represses
the activity of Hedgehog- and Wingless-signaling pathways. It remains to be
seen whether Notch activity in Drosophila follicle cells impinges
directly on the transcriptional regulation of string, dacapo and
fzr or whether Notch acts through another signaling pathway.
Our studies of follicle cell cycle programs in the ovary are important because they provide a comparison with other cell cycle programs in Drosophila development. Furthermore, the transition from mitotic cycles to endocycles is a universal phenomena; understanding how molecular events bring about this transition in follicle cells will shed light on such transitions in general.
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Department of Cell Biology, Duke University Medical
Center, Durham, NC 27710, USA
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