Research article 3169

The mitotic-to-endocycle switch in Drosophila follicle cells is

executed by Notch-dependent regulation of G1/S, G2/M and M/G1
cell-cycle transitions
Halyna R. Shcherbata*, Cassandra Althauser*, Seth D. Findley† and Hannele Ruohola-Baker‡
Department of Biochemistry, University of Washington, J591, HSB, Seattle, WA 98195-7350, USA
*These authors contributed equally to this work
†Present address: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
‡Author for correspondence (e-mail: [email protected])

Accepted 16 March 2004

Development 131, 3169-3181

Published by The Company of Biologists 2004
doi:10.1242/dev.01172

Summary
The Notch signaling pathway controls the follicle cell overexpression of the growth controller Myc does not
mitotic-to-endocycle transition in Drosophila oogenesis by induce premature endocycles but accelerates the kinetics of
stopping the mitotic cycle and promoting the endocycle. To normal endocycles. We also show that Archipelago (Ago),
understand how the Notch pathway coordinates this a SCF-regulator is dispensable for mitosis, but crucial for
process, we have identified and performed a functional endocycle progression in follicle epithelium. The results
analysis of genes whose transcription is responsive to the support a model in which Notch activity executes the
Notch pathway at this transition. These genes include the mitotic-to-endocycle switch by regulating all three major
G2/M regulator Cdc25 phosphatase, String; a regulator cell cycle transitions. Repression of String blocks the M-
of the APC ubiquitination complex Hec/CdhFzr and an phase, activation of Fzr allows G1 progression and
inhibitor of the CyclinE/CDK complex, Dacapo. Notch repression of Dacapo assures entry into the S-phase. This
activity leads to downregulation of String and Dacapo, and study provides a comprehensive picture of the logic that
activation of Fzr. All three genes are independently external signaling pathways may use to control cell cycle
responsive to Notch. In addition, CdhFzr, an essential gene transitions by the coordinated regulation of the cell cycle.
for endocycles, is sufficient to stop mitotic cycle and
promote precocious endocycles when expressed Key words: Drosophila, Notch, String, Fizzy related, Dacapo, Cell
prematurely during mitotic stages. In contrast, cycle, Follicle cells

Introduction induce phosphorylation events that control whether the cell

Cancer cells often show abnormal cell cycle control in response enters the M- or S-phase. CycA/Cdk1 and CycB/Cdk1
to external stimuli and this feature has attracted much scientific complexes that can be activated by Cdc25-type phosphatases
interest. Many examples now exist in which signaling mediate M-phase control. S-phase is controlled by other
pathways can induce the G1/S cell cycle transition. Fewer complexes: CycE/Cdk2, CycA/Cdk2, and CycD/Cdk4 or
examples exist whereby signaling pathways stop cells from Cdk6. Some of these complexes are known to be negatively
cycling and dividing. Studies on a natural variation of the regulated by several Cdk inhibitors, such as p27Kip1. p27Kip1
mitotic cycle, the endocycle, have helped to illuminate this inhibits CycE/Cdk2 complexes and thereby arrests cells in the
question (Edgar and Orr-Weaver, 2001). The endocycle (as G1-phase (Olashaw and Pledger, 2002).
seen for example in cardiomyocytes, trophoblasts, and Cells normally progress through the cell cycle in a series of
Drosophila nurse and follicle cells) is a variation of the normal sequential steps in which each step is dependent on the proper
cell cycle in which rounds of DNA replication and growth completion of the previous stage. However, endocycling cells
occur without intervening mitoses (Zimmet and Ravid, 2000). are exceptions to this rule because they proceed to S-phase
A critical characteristic of the endocycle is its capacity to without completing M-phase. How this occurs is not
bypass the important controls that regulate normal transitions understood. In some endocycling cells, bypassing the mitotic
in mitotic cycles. How this bypass is accomplished is largely stage correlates with eliminating or reducing the Cdk activity
unknown; although some of the factors that control the required for M-phase entry. For example in Drosophila early
endocycle have been partially characterized (Edgar and Orr- embryonic cells and follicle cells, components and regulators
Weaver, 2001). Importantly, a comparative approach that of the M-phase can be eliminated at a transcriptional level
addresses this question might aid our understanding of how and/or at a posttranscriptional level (Deng et al., 2001; Sauer
cells become cancerous. et al., 1995; Sigrist and Lehner, 1997). Posttranscriptional
In normal mitotic cells cyclin-dependent kinases (Cdks) modulations are often mediated by ubiquitination via the APC-
3170 Development 131 (13) Research article

complex with degradation of the cyclins and securins (Peters, amorphic allele of Delta that is produced by excision of the promoter
2002; Vodermaier, 2001). region, transcription start site and first exon) (Heitzler and Simpson,
The key question in endocycle regulation is how the 1991; Zeng et al., 1998); Su(H) FRT40A [Su(H)SF8 is a strong loss-
transition from the mitotic phase to the endocycle is controlled. of-function but not a null allele of Suppressor of hairless, a gift from
Two signaling pathways have been identified as regulators of S. Blair), whereas Su(H)047 FRT40A is a null allele] (Li et al., 1998);
fzrie28 FRT101 (Schaeffer et al., 2004); FRT42B dap4 [dap4 is a w–
the mitotic-to-endocycle transition: the thrombopoetin pathway, derivative associated with 95%-100% lethality, which was found to
which acts during differentiation of megakaryocytes and the represent an imprecise excision that resulted in an intragenic deletion
Notch pathway, which acts during Drosophila oogenesis and of the dacapo gene] (Lane et al., 1996), ago1 FRT80B, ago3 FRT80B
during the differentiation of trophoblasts (Deng et al., 2001; and ago4 FRT80B all have similar archipelago phenotypes (Moberg
Lopez-Schier and St Johnston, 2001; Nakayama et al., 1997; et al., 2001). For generating follicle cell clones we used: hsFLP;Ubi-
Wu et al., 2003; Zimmet and Ravid, 2000). Human GFP FRT40A, hsFLP;Ubi-GFP FRT42B, hsFLP;lacZ FRT42B,
teratocarcinomas also seem to arise from defects in the mitotic- hsFlp;;Ubi-GFP FRT80B, hsFlp;;FRT82B Ubi-GFP, yw Ubi-GFP
to-endocycle transition in trophoblasts (Cross, 2000). The key FRT101, w–;;MKRS P{ry=hsFlp}86E/Tm6BTb, hsFlp;;UAS-
cell-cycle targets of these pathways, however, remain elusive. GFPact<FRT-CD2-FRT<Gal4/TM3 (Pignoni and Zipursky, 1997).
In Drosophila follicle cells the function of the Notch pathway For analysis of overexpression patterns the following stocks were
used: UAS-stgN4 and UAS-stgN16 (Bloomington Stock Center/Bruce
in the mitotic-to-endocycle transition has been well established Edgar), UAS-inx2 (Stebbings et al., 2002), mew∆.Scer\UAS (Li et al.,
(Deng et al., 2001; Lopez-Schier and St Johnston, 2001). 1998), w–;UAS-cycD/CyOGFP;Dichaetae/TM6B, UAS-cycE, UAS-
Specifically, the ligand Delta is secreted by germ line cells and cycA, UAS-dap II.2 and UAS-dap II.3 (gifts from Bruce Edgar), UAS-
activates Notch in the follicle cells. Subsequently, the fzr III.2, UAS-fzr II.1, fzrie28 (gifts from Christian Lehner) and w;
cytoplasmic portion of Notch is cleaved by Presenillin and UAS-dMyc132, a gift from Robert Eisenman (Johnston et al., 1999).
moves to the nucleus where, in combination with a transcription We also used the following p53 lines: w[1118];P{w[+mC]=GUS-
factor, Suppressor of Hairless [Su(H)], it affects the p53}2.1, dominant negative constructs of p53 w[1118];P{w[+mC]=
transcription of various target genes. Lack of Notch activity in GUS-p53.259H}3.1 and w[1118];P{w[+mC]=GUS-p53.Ct}3.1/
Drosophila follicle cells leads to prolonged mitosis at the TM6B, Tb[1] (Bloomington Stock Center/Gerry Rubin), and the p53
expense of endocycles. This has led to the suggestion that Notch viable mutants y[1]w[1118];p53[5A-1-4] and y[1]w[1118];p53[11-
1B-1] (Bloomington Stock Center/Kent Golic). Rbf120a/FM7 and
functions in this context as a tumor suppressor (Deng et al., Rbf14/FM7 were gifts from Giovanni Bosco and Terry Orr-Weaver. In
2001; Lopez-Schier and St Johnston, 2001). Because very few addition, we used the fzr-lacZ fusion line G0326 (Bloomington Stock
signaling pathways have been identified that stop the mitotic Center), 6.4-string-lacZ fusion transgene construct [a gift from Bruce
cell cycle, it is important to understand in detail the relationship Edgar (Lehman et al., 1999)] and various dap transgene constructs
between the Notch pathway and known cell cycle regulators. [gifts from Christian Lehner (Meyer et al., 2002) and Harold Vaessin
We have previously shown that one of the cell cycle (Liu et al., 2002)]. dap-lacZ fusion transgene constructs that lacked
components that responds to Notch activity at the the 1.5 kb upstream region of the dap gene gave no staining, however,
transcriptional level, String/Cdc25 phosphatase, which is a most of the dap-gm constructs, which included the entire gene as well
regulator of the transition between S- and M-phase, is not as different lengths of the promoter region fused to a myc-epitope tag,
sufficient, by itself, to keep all the cells in mitotic phase, showed clear staining in the follicle cells before stage 6 and a
downregulation of expression thereafter.
suggesting that other components are needed for the mitotic-
to-endocycle transition (Deng et al., 2001; Schaeffer et al., Generation of follicle cell clones
2004). One such Notch-regulated component, essential for Drosophila melanogaster stocks were raised on standard cornmeal-
the mitotic-to-endocycle transition is Hec1/CdhFzr, a WD40- yeast-agar-medium at 25°C. To obtain follicle cell clones, 1-to-5-day-
domain regulator of the APC-Ubiquitination complex. old flies were heat-shocked as adults for 50-60 minutes at 37°C and
Hec1/CdhFzr, which acts with other cellular components put in freshly yeasted vials for 3 or 5 days. To obtain germ line clones,
important for M-phase entry, is dispensable for mitosis but flies were heat-shocked as second and third instar larvae for 2 hours
essential for the mitotic-to-endocycle transition in follicle cells on 2 consecutive days. Once they emerged as adults, they were placed
(Schaeffer et al., 2004). However, cells in fzr–/– clones do not in vials with fresh yeast paste for 1-5 days prior to dissection.
prolong mitosis unless accompanied with ectopic string. Nuclear preparation and flow cytometric analysis
We have now shown that this APC regulator is sufficient to Nuclear preparation was done essentially as described in Bosco et
induce, albeit with low penetrance, premature endocycles if al. (Bosco et al., 2001) and Calvi and Lilly (Calvi and Lilly, 2004)
precociously expressed in the follicle cell epithelium. We with minor modifications. Ovaries from 50-100 females were
furthermore identify an inhibitor of the CyclinE/CDK incubated in 1 ml of 5 mg/ml collagenase (Blend type H, Sigma-
complex, Dacapo, that is reduced because of Notch activity in Aldrich C8051) in 90% Graces insect medium at 4°C for 15 minutes
the mitotic-to-endocycle transition as a repressor of endocycles on a Clay-Adams Nutator. Ovaries were then disrupted by pipeting
in follicle cells. Notch activity, therefore, executes the mitotic- them several times through a P-1000 tip and then pelleted in an
to-endocycle transition by regulating three cell cycle Eppendorf centrifuge by a 2-second spin at 1000 g. The pellet was
transitions: repression of String blocks M-phase, activation of resuspended in 500 µl of buffer A (15 mM Tris-HCl, pH 7.4, 60 mM
Hec1/CdhFzr allows G1 progression, and repression of Dacapo 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-
assures entry into S-phase. 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
Materials and methods resulting lysate was cleared by serial passage through 150-, 100-,
50- and finally 30-µm Nitex filters (Sefar America, Depew, NY,
Fly stocks USA). Nuclei were then pelleted by passage through a sucrose (0.25
The following fly stocks were used: FRT82B Dlrev10e (Dlrev10 is an M-2.5 M, in 1X Buffer A) density step gradient 15,000 g for 20
 Notch regulates cell cycle transitions 3171

minutes at 22°C in a Beckman TLS-55 rotor and resuspended in 200 Studying the Cyclin D role in mitotic-to-endocycle
µl of buffer A that contained 0.1% NP-40 and 20 µg/ml propidium transition
iodide. Follicle cells nuclear ploidy was determined by fluorescence- We analyzed the expression pattern of CycD during mitotic-to-
activated cell sorting (FACS) analysis using a Becton Dickinson endocycle transition, because it has been shown to be critical for cell
FACScan cytometer by numbering the intensity of propidium growth in Drosophila (Chen et al., 2003; Datar et al., 2000; Meyer et
fluorescence in stained nuclei. Nuclei were exited with a 488 nm al., 2000) and the cell growth is a major component of endocycle.
laser, and the emission was monitored through a 585/42 nm band Surprisingly, a clear downregulation of cycD RNA level was observed
pass filter. Results were analyzed by using CellQuest and Multicycle at the onset of endocycles, stage 6 (data not shown). The functional
software. relevance of this downregulation is not clear because overexpression
of the protein, in combination with its kinase Cdk4, does not
Staining procedures dramatically affect entry into endocycles. During these
Ovaries were dissected in phosphate-buffered saline (PBS) and fixed overexpression studies, we observed no difference in the size of nuclei
while shaking on a nutator for 10 minutes in PBS containing 5% compared with those in wild-type cells and there was no upregulation
Formaldehyde. Next, they were rinsed with PBT (PBS/0.2% Triton of CycB or PH3 (data not shown).
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 Results
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 The endoreplication cell cycle or endocycle is a variation of
temperature. The ovaries were then incubated in secondary the normal mitotic cell cycle, in which cells increase their
antibodies overnight at 4°C. The next day they were rinsed with PBT genomic DNA content without dividing. How the transition
(4×15 minutes) and stained with DAPI (1 µg/ml in PBT) for 10 from the mitotic cycle to the endocycle is regulated is not well
minutes. Finally, they were washed with PBT twice four times (5 understood. Drosophila oogenesis provides an excellent
minutes, each wash) and dissected onto slides in 70% glycerol, 2% system in which to study this transition (Calvi et al., 1998).
NPG, 1X PBS. In the Drosophila ovary both the germ line and somatic cells
Follicle cells were labeled with BrdU as described previously arise from stem cell populations located in an anterior ovary
(Bosco et al., 2001; Calvi and Lilly, 2004; Lilly and Spradling, 1996) structure called the germarium (Fig. 1A). At the posterior end
with slight modifications. Ovaries were dissected in Grace’s insect
medium and then incubated with 10 µM BrdU (Boehringer
of the germarium, somatic cells encapsulate a 16-cell cyst
Mannheim) in the same medium for 1.5 hours at room temperature. of germ line cells, a configuration in which the oocyte
The ovaries were then fixed in 10.5% formaldehyde for 15 minutes, will eventually develop during a three-day period (this
washed with PBT, then treated with 2N HCl for 45 minutes. Sodium developmental process has been divided into 14 stages).
borate (100 mM) was used for neutralization. The tissues were then Meanwhile, the somatic follicle cells will undergo three tightly
rinsed with PBT three times (10 minutes, each rinse) and blocked in developmentally controlled cell cycle modifications (Calvi et
PBTB for half an hour at room temperature and incubated with mouse al., 1998). First, these epithelial cells undergo a mitotic division
anti-BrdU antibodies (Becton Dickinson) overnight at 4°C. The next program that gives rise to approximately 1000 follicle cells by
day, ovaries were rinsed with PBT six times (5-10 minutes, each stage 7 in oogenesis (Fig. 1A, part I). At this mid-oogenesis
rinse), blocked in PBTB for 30 minutes at room temperature and point, signaling through the Notch pathway stops the mitotic
incubated with secondary antibodies for 2 hours at room temperature.
Thereafter, the ovaries were rinsed with PBT four times (15 minutes,
cycles in the follicle cells and allows them to enter
each rinse) and stained with DAPI for 10 minutes. Finally, they were endoreplication to become polyploid (Deng et al., 2001;
washed with PBT twice (5 minutes, each wash) and dissected onto Lopez-Schier and St Johnston, 2001) (Fig. 1A, part II). After
slides in 70% glycerol, 2% NPG, 1X PBS. stage 6, the follicle cells then undergo three endocycles to
Confocal microscopy, X-gal staining and in situ hybridization were become polyploid. Later in oogenesis at stage 10B in response
performed as described previously (Keller Larkin et al., 1999; to unknown developmental signals, four different loci,
Tworoger et al., 1999). For in situ RNA hybridization studies stg encoding several different genes two of which are chorion
cDNA (LD47579) was labeled with fluorescine, whereas cycD genes, synchronously initiate a gene amplification event, that
(LD22957), cycE (LD22682), cycA (LD44443), cycB (LD23613), and increase their copy number (Fig. 1A, part III). During this
fzr (LD21270) cDNAs were labeled with digoxigenin (all cDNAs phase all other genomic replication origins remain inactive
were from the Berkeley Drosophila UniGene Collection). A two-
photon laser-scanning confocal microscope (Leica TCS SP/MP) was
(Orr-Weaver, 1991; Spradling, 1999). The chorion genes
used in this study. encode the eggshell proteins and amplification of these genes
The following primary antibodies were used at the designated is needed to produce sufficient chorion protein for a normal
dilutions: mouse anti-Fasciclin III (1:20), mouse anti-CycA (1:20) eggshell. These three replication patterns are readily observed
and mouse anti-CycB (1:20) from Developmental Studies by BrdU analysis (Calvi et al., 1998) (Fig. 1B). In addition,
Hybridoma Bank, rabbit anti-CycA [1:100, David Glover (this these cell cycle programs can be distinguished by different
antibody showed CycA downregulation at stage 6 but some markers, for example, CycB and PH3 (Deng et al., 2001) (Fig.
immunoreactive material was later observed at stage 8)], mouse anti- 1C).
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, Hec1/CdhFzr is sufficient to stop mitosis and induce
Upstate Biotechnology), rabbit anti-Fizzy-related (1:800, a gift from premature endocycles
Christian Lehner), mouse or rabbit anti-β-gal (1:5000, Sigma) and
mouse anti-c-Myc (1:50, Calbiochem). The following secondary We have previously described the role of Notch signaling in
antibodies were used at the designated dilutions: Alexa 488, 568 or downregulating string at stage 6 of oogenesis to allow the cells
633 goat anti-mouse (1:500), Alexa 488, 568 or 633 goat anti-rabbit to transit into the endocycle (Deng et al., 2001) (Fig. 1D). In
(1:500, Molecular Probes), Alexa 488 goat anti-guinea pig (1:500, order to find other cell cycle genes activated by Notch activity
Molecular Probes). after/during the transition from mitotic-to-endocycle, we
3172 Development 131 (13) Research article

performed an expression screen for genes differentially molecules, transcriptional control proteins and cell cycle
expressed before and after the transition. We screened 400 regulators (Table 1). The two cell cycle regulators, Fizzy
lethal X-chromosome P-element enhancer trap-lines (Peter et related (Hec1/CdhFzr) and Myc (Bourbon et al., 2002), were
al., 2002) for changes in expression levels at stage 7 using the analyzed in more detail. The expression of string is observed
β-gal reporter gene. Seven genes that fall into three interesting until stage 6, whereas high fizzy related (fzr) expression marks
functional groups were obtained from this screen: adhesion the start of endocycles (Fig. 1D,E). Importantly, we have
shown that Hec1/CdhFzr is required for the
mitotic-to-endocycle transition in these cells
(Schaeffer et al., 2004). Furthermore, we have
shown that the combination of reduced
Hec1/CdhFzr and prolonged Stg expression
can keep the follicle cells in the mitotic cycle
past stage 6.
To test what is required to turn mitotic
cycles to endocycles prematurely, we over-
expressed candidate genes at stages 1-6
and analyzed the effects in follicle cells
undergoing mitotic cycles. Premature
expression of the adhesion molecules did not
alter the timing of the mitotic-to-endocycle
transition (Table 1). However, premature
expression of the Notch-responsive cell
cycle component, Hec1/CdhFzr, caused the
formation of enlarged nuclei, a potential
indication of precocious endocycles (Fig.
2A,B) (Schaeffer et al., 2004). Importantly,
analysis of cell cycle markers revealed a
premature reduction of cyclins A and B (70%
and 91% reduction, respectively; Fig. 2C).
Consequently, a halt in mitotic cell cycle (60%
reduction in PH3 staining; Fig. 2C) was
observed because of the premature expression
of Hec1/CdhFzr. In contrast, as a control we
overexpressed other cell cycle components,
such as dMyc and p53 and observed no change
in the expression of mitotic markers compared
with that seen in wild-type cells (Fig. 2C).
These findings suggest that the premature
expression of Hec1/CdhFzr is sufficient, at
least partially, to stop mitosis.
The enlarged nuclei observed upon
overexpression of Hec1/CdhFzr could be
caused via a ploidy-independent mechanism
(Gao and Pan, 2001; Tapon et al., 2001) or via
increase in ploidy as some cells undergo
ectopic endocycles. To distinguish between
these possibilities, we determined the exact
DNA content in the cells overexpressing fzr by
FACS on purified ovarian nuclei (Bosco et al.,
Fig. 1. Cell cycle transitions in epithelial follicle cells. (A) Follicle cells in Drosophila 2001; Calvi et al., 1998; Lilly and Spradling,
oogenesis undergo two cell cycle transitions: from a mitotic cell cycle to an endocycle 1996). Although control nuclei revealed the
and from endocycle to amplification (A, parts I-III, drawing of the stages of oogenesis). normal 2n, 4n, 8n and 16n DNA peaks, some
From the germarium (g) to stage (st) 6, somatically derived follicle cells undergo of the nuclei from UAS-fzr mutants showed
mitotic cycles, which are not synchronized (A, part I). At stage 7 they switch to DNA content of 32n, comparable with that in
endocycles. From stage 7 to stage 10A, these cells undergo three rounds of
Rbf mutants, in which follicle cells are known
endoreplication (A, part II) and thereafter switch to the localized replication pattern
characteristic of chorion gene amplification (A, part III). These three replication to undergo extra endocycles (Bosco et al.,
patterns are observed in BrdU incorporation analysis (B). The staining of mitotic 2001) (Fig. 2H-J). These data suggest that
markers Cyclin B (CycB) (red arrows), Phospho-Histone 3 (PH3) (green arrows) (C) some of the follicle cells overexpressing fzr
and stg (6.4) promoter construct (D) show expression in mitotic follicle cells, whereas not only stopped mitotic cycles prematurely
Fzr enhancer trap line, fzrG0326 (β-gal expression, red arrow) shows Fzr expression in but also proceeded through extra endocycles
endocycling cells (E). Green, PH3; red, CycB (C); Anti-β-gal (D,E); blue, DAPI (C-E). increasing the ploidy of the cells.
 Notch regulates cell cycle transitions 3173

Table 1. Genes that show a change in expression levels at the mitotic-to-endocycle transition in follicle cells
Cell cycle phenotypes
Functional group Protein Expression Loss of function Ectopic expression
Adhesion Fasciclin 2 After stage 7-8 N/A N/A
Innexin 2 After stage 7-8* N/A No phenotypes (this study)
PS1α (mew) After stage 7-8 N/A No phenotypes (this study)
Transcriptional control BRC After stage 7-8 Abnormal amplification (Tzolovsky et al., Abnormal amplification (Tzolovsky et al.,
1999) 1999)
Trf2 After stage 7-8 N/A N/A
Cell cycle control Fzr After stage 7-8 Defective endocycle (Schaeffer et al., 2004) Precocious endocycle (this study)
Myc After stage 7-8 Defective endocycle (Maines et al., 2004) Accelerated endocycle (this study)

*mRNA expression pattern of innexin 2 in Drosophila follicle cells has been previously described (Stebbings et al., 2002).

Fig. 2. Fzr is partially sufficient to induce

precocious endocycles, dMyc to
accelerate endocycles. Ectopic expression
of fzr (hsFlp; UAS-fzr; UASGFP
act<FRT-CD2-FRT<Gal4) reduces CycA
and B levels, stops mitosis (A,B) and
allows the formation of large nuclei (B)
indicative of precocious endocycles prior
to stage 6 in oogenesis. The diagram in C
presents the percentage of the wild-type
and mutant follicle cells that show
positive staining to mitotic markers
during the mitotic stage. fzr (D,F,J) and
myc (E,G,K) overexpression (hsFlp; UAS-
fzr or UAS-myc; UASGFP act<FRT-CD2-
FRT<Gal4) generate enlarged nuclei,
which is indicative of abnormal
endocycles but do not affect the
amplification stage because normal BrdU-
incorporation pattern at stage 10B-12 (red
arrows) is observed in cells
overexpressing fzr (F) and myc (G). (H-K)
FACS profiles of DNA content in UAS-fzr
(J) and UAS-myc (K) mutant nuclei
compared with these in WT nuclei (H)
show the appearance of a cell population
that has 32n DNA, also observed in Rbf
mutants (I) (Bosco et al., 2001). Green,
GFP (A,D,E), Fzr Ab (F); red, CycB (A),
DAPI (D,E), BrdU (F,G); blue, DAPI
(A,F,G).
3174 Development 131 (13) Research article

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 Fig. 3. The Cyclin E levels are dynamic during cell cycle transitions;
these cells will enter premature endocycles (Fig. 2B,C). constant CycE levels halt endocycles and amplification. (A) Wholemount
One possible explanation for these results is that in situ hybridization indicates equal level of cyclin E mRNA in the follicle
components at G1/S transition, such as G1 cyclins, cells throughout oogenesis (arrows). (B,C) Cyclin E antibody staining
further restrict from entering the premature endocycles pattern (green) between stages 5 (B) and 8 (C) shows a clear
by blocking entry into the S-phase in mitotic cells. downregulation of the protein after the transition to the endocycle, when
In other systems G1/S transition is controlled in part cells stop expressing PH3 (red). Cells overexpressing CycE (hsFlp;;UAS-
by factors that regulate cell growth such as CycD or Myc cycE/UASGFP act<FRT-CD2-FRT<Gal4) express mitotic cyclins B and A
(Frei and Edgar, 2004). Curiously, CycD does not seem past stage 6 in oogenesis (D-E, arrow), have smaller nuclei (F,F′) and show
highly reduced BrdU incorporation during endocycling (G,G′) and
to be critical for endocycles in follicle cells (see
amplification (H,H′) stages, suggesting that cells ectopically expressing
Materials and methods). This presents a paradox because cyclin E fail to undergo S-phase and are halted at G2. Green, CycE (B,C),
CycD is known to be critical for cellular growth in GFP (D-F), BrdU (G,H); red, PH3 (B,C), CycB (D), CycA (E), CycE (F-
Drosophila (Datar et al., 2000; Meyer et al., 2000) and H); blue, DAPI.
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 were indicative of extra endocycles because cells with 32n
to this apparent paradox, because this screen demonstrated ploidy were observed among these follicle cells (Fig. 2K).
that Myc, another component required for growth, is Similar to the case of fzr overexpression, BrdU incorporation
transcriptionally upregulated at the transition. revealed normal amplification patterns during stage 12 in
dMyc is the Drosophila homologue of the BHLHZ Myc- oogenesis in cells overexpressing myc (Fig. 2G), suggesting
oncogene family of transcription factors. Recent studies have that the extra endoreplication observed during myc
shown that dMyc has a function in cellular growth (Iritani and overexpression was not caused by defects in the endocycle-
Eisenman, 1999; Johnston and Gallant, 2002; Johnston et al., to-amplification transition. Because the higher ploidy
1999). To test whether dMyc can influence the mitotic-to- accompanies no change in the timing of mitotic-to-endocycle
endocycle transition, we overexpressed dmyc in developing or endocycle-to-amplification transition, we conclude that this
follicle cells and analyzed cell cycle markers in both cell cycle defect was caused by faster endocycle kinetics than in wild-
programs. No obvious change was observed during the mitotic type follicle cells. Thus, these data suggest that Myc-dependent
stage upon ectopic expression of dmyc (Fig. 2C); however growth can regulate endocycle kinetics in follicle epithelial
during the endocycling stage, cells overexpressing dmyc were cells.
larger than the neighboring wild-type cells (Fig. 2E). In
particular, larger nuclei were observed in 32% and 55% of the Proper CycE regulation is required for the mitotic-to-
cells at stage 7-9 and 12 (respectively) after overexpressing endocycle transition
dmyc (Fig. 2B). FACS analysis revealed that these large nuclei Another G1/S-regulator that might play a role in the proper
 Notch regulates cell cycle transitions 3175

Table 2. Proper Cyclin E regulation is crucial for endocycles: increased Cyclin E activity blocks the cells in G2, reduced
Cyclin E in G1
Cell cycle phenotypes at stage 7-12 follicle cells*
Prolonged staining of mitotic markers BrdU incorporation
Small nuclei
Genotype (lack of endocycles) (CycA,B) (CycE) PH3 Endocycles† Amplification Cell cycle arrest
hsFlp;; UAS-cycE/UASGFP act + 63% + 44% + – – – G2
<FRT-CD2-FRT<Gal4
hsFlp: ago FRT80B/Ubi-GFP + 92% + 37% + 91% – – – G2
FRT80B
hsFlp; UAS-dap;UASGFP act + 71% – + activity (–) – – – G1
<FRT-CD2-FRT<Gal4
Wild type – – – – + + No arrest

*n>100 cells for each experiment

†Although in wild type, 38% of all endocycling follicle cells are BrdU positive, in mutant clones only 6-7% of the cells showed BrdU staining.

mitotic-to-endocycle transition in follicle cells is Cyclin E et al., 2000; Lane et al., 1996) and Archipelago, a recently
(Calvi et al., 1998). Drosophila Cyclin E forms a complex with characterized F-box, WD repeat protein found to target CycE
the DmCdc2c/Cdk2 kinase and controls the progression for SCF-dependent degradation (Moberg et al., 2001;
through the S phase; its downregulation limits embryonic Strohmaier et al., 2001).
proliferation and its oscillation is required for endocycling
(Follette et al., 1998; Knoblich et al., 1994; Weiss et al., 1998). Dacapo downregulation by Notch signaling at
The cycE mRNA expression pattern in the follicle cells during mitotic-to-endocycle transition is required for proper
oogenesis reflects a continuous requirement both in mitotic and endocycling
endocycles without downregulation at the mitotic-to-endocycle The Cip/Kip families of cyclin-dependent kinase inhibitors
transition; but rather, an equal level of ‘patchy’ cycE mRNA (CKI) are the main effectors linking developmental programs
levels within the follicle cells throughout oogenesis (Fig. 3A). and cell cycle progression (Liu et al., 2002). Drosophila
The same ‘patchy’ pattern of CycE protein level is seen in dacapo (dap) encodes an inhibitor of cyclin E/cdk2 complexes
mitotic and endocycling follicle cells, indicating the cell cycle- with similarity to the vertebrate Cip/Kip inhibitors (Lane et al.,
dependent oscillation of CycE protein. However, we observed 1996; Liu et al., 2002; Meyer et al., 2002) and thereby inhibits
a clear, but not complete, downregulation of CycE protein entry into the S-phase. In accordance with this role,
levels at approximately stage 7, just after the follicle cells mammalian family members are upregulated during
stopped expressing CycB and PH3 (mitotic markers) (Fig. checkpoint activation and are expressed in terminally
3B,C). In follicle cell clones mutant for a transcription factor differentiated tissues. Dacapo contains a conserved amino-
in the Notch pathway, Su(H), the CycE levels were somewhat terminal domain that binds to and inhibits all kinases involved
abnormally regulated during endocycles, which suggested a in G1/S transition. This inhibition is achieved by a
role for the Notch signaling pathway in this posttranscriptional conformational change in the cyclin/Cdk complex (Pavletich,
regulation (data not shown). 1999).
The observed change of CycE level in the mitotic- In follicle cells, the in situ mRNA hybridization pattern for
to-endocycle transition is critical because continuous dacapo indicates that its transcriptional level is downregulated
overexpression of CycE leads to abnormalities in the mitotic- at stage 6-7 (Fig. 4A), suggesting a potential regulation by the
to-endocycle transition; the cells of clones overexpressing cycE Notch pathway. Because dacapo has previously been shown
exhibit prolonged expression of CycA and CycB and have to be developmentally controlled by an extensive promoter
smaller nuclei than their neighbours (44% and 63%, region, we used different dap-constructs (Liu et al., 2002;
respectively; n=214) (Table 2, Fig. 3D-F) (Lilly and Spradling, Meyer et al., 2002) and studied their expression patterns in the
1996). However, no PH3 staining is observed among these follicle cells during oogenesis. The smallest constructs, that
mutant cells after stage 6, suggesting that they do not undergo faithfully represent dacapo mRNA pattern (dap5gm and
prolonged mitosis. Likewise, BrdU incorporation is inhibited dap6gm), include the entire gene as well as 1.8 kb and 2 kb
in these cells after stage 6 (including the amplification stage as of the promoter region fused with a myc-epitope tag. These
shown before by Calvi et al. (Calvi et al., 1998), implying that transgenic lines showed clear myc-epitope staining in the
they also fail to undergo S-phase (Fig. 3G,H). Taking into follicle cells before stage 6 and a downregulation of expression
account these findings and the upregulation of CycB and CycA, thereafter (Fig. 4B). One of these constructs, dap5gm, was used
but lack of PH3 staining, we hypothesize that the cells as a marker for Dacapo expression in the following studies.
overexpressing CycE are halted at the G2 stage of the cell cycle In order to determine whether Notch signaling controls the
in the mitotic-to-endocycle transition. downregulation of dacapo at stage 6, we analyzed Dacapo
These and other data (Calvi et al., 1998; Lilly and Spradling, expression in follicle cells surrounding Delta mutant germ line
1996; Sauer et al., 1995) suggest that correct CycE protein clones in a fly that contained the dap5gm construct. In the Dl
levels are critical for the proper mitotic-to-endocycle germ line clones, we clearly observed a prolonged expression
transitions. We therefore turned our investigation to the role of dap5gm in stages later than 6 (Fig. 4C), suggesting that the
of two regulators of CycE protein: Dacapo, a CIP/KIP transcriptional downregulation of dacapo is dependent on
homologous protein found to inhibit CycE activity (de Nooij Notch activity.
3176 Development 131 (13) Research article

To analyze whether the Notch-dependent repression of attempts to enter endocycles and precocious expression of
dacapo was important for the mitotic-to-endocycle transition, Hec1/CdhFzr (Fig. 2A,C).
we tested the functional consequence of dacapo loss-of-
function and prolonged expression for follicle cell cycle Ago is essential for endocycles but dispensable for
control. Dacapo loss-of-function clones revealed no obvious mitotic cycles
phenotype; nuclei sizes were normal and endocycle did not Because dacapo is downregulated after the mitotic-to-
appear to be inhibited. Overexpression of dacapo has endocycle transition, we investigated whether archipelago, a
previously been shown to inhibit entry into late amplification second regulator of CycE protein level was required for proper
(Calvi et al., 1998) and, in the salivary gland, overexpression oscillations of CycE during endocycles. Archipelago is an F-
of dacapo inhibits endoreplication (Weiss et al., 1998). We box protein with seven tandem WD repeats that recognizes
likewise observed cell cycle defects because of the prolonged auto-phosphorylated CycE. Ago protein binds directly to
expression of dap: smaller nuclei and a failure to incorporate Cyclin E and targets it for ubiquitin-mediated SCF-dependent
BrdU, indicating a lack of S-phases (Fig. 4D-F). CycA and degradation (Moberg et al., 2001; Strohmaier et al., 2001). The
CycB, although upregulated in cells overexpressing CycE past in situ hybridization pattern of archipelago reveals continuous
stage 6, were not upregulated in cells overexpressing dap mRNA expression both in the mitotic and endocycling stages
(Table 2). These results suggest that Notch-based (Fig. 5A). To investigate the role of Archipelago in these cells
downregulation of dacapo is critical for endocycling because we made follicle cell clones with three different ago alleles
overexpression of dacapo, a CIP/KIP-type cyclin-dependent (Moberg et al., 2001). As expected, we saw an increase in CycE
kinase inhibitor of Cdk2/CycE complexes, can halt the follicle protein in most mutant follicle cells at all stages during
cell endocycles at the apparent G1/S transition. Also, one oogenesis in all three mutants (Fig. 5B). We then analyzed the
possibility is that Dacapo in mitotic cells aborts premature 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).
However, lack of Ago activity seemed to be
detrimental for entering the endocycle: ago clones
showed much reduced or no BrdU incorporation and
small nuclei size after stage 7 (Fig. 5F,G, Table 2). In
addition, CycB expression is prolonged in 37%
(n=200) of ago-mutant follicle cells past stage 6 (Fig.
5E). We also analyzed CycA, a marker of the S and G2
phase, and observed an elevation in ago clones (data
not shown). At the later stages no amplification was
observed and some of the mutant cells were seemingly
apoptotic (Fig. 5H and data not shown). The
phenotypes in ago-clones (elevation of CycA and
CycB levels, small nuclear size, lack of endocycle and
amplification and some cell death at late stages) are
also observed upon overexpression of CycE and were
Fig. 4. Notch-dependent repression of the Cyclin E inhibitor Dacapo is indicative of a G2 block (Fig. 3D-H, Table 2). Because
required for mitotic-to-endocycle transition. (A) The dacapo mRNA in situ overexpression of CycE is observed in most cells
pattern indicates transcriptional downregulation after transition to endocycles mutant for ago, we conclude that the phenotypes
(light arrow, st. 7). (B) A similar expression pattern was observed in a observed during the mitotic-to-endocycle transition in
dap5gm construct, which includes the entire gene and a part of the promoter ago clones could be because of the high level of CycE
region fused with myc-epitope tag (arrow indicates follicle cells staining activity. These data suggest that an Archipelago/SFC-
prior to st. 7). (C,C′) Prolonged follicle cell expression of dap5gm in st. 9 dependent regulation of CycE protein levels is
Delta germ line clone shows that dap is controlled by Notch signaling. (D) dispensable for mitotic divisions but essential for
Overexpression of dap (hsFlp; UAS-dap; UASGFP act<FRT-CD2-
FRT<Gal4) inhibits endoreplication: follicle cells overexpressing dacapo
endocycles in follicle epithelium.
have smaller nuclei (D′) than the neighboring wild-type cells and they fail to
incorporate BrdU during both the endocycling (E,E′) and the amplification
Notch controls independently String,
(F,F′) stages, indicating that these cells could not undergo the S-phase and Hec1/CdhFzr and Dacapo expression
probably are blocked at the G1/S transition. Green, GFP (C,D), BrdU (E,F); Notch activity affects the expression of Stg (Deng et
red, CycE (D), Dap (E,F); blue, DAPI. al., 2001), Fzr (Schaeffer et al., 2004) and Dacapo (this
 Notch regulates cell cycle transitions 3177

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
other’s 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.
Premature expression of Hec1/CdhFzr blocks
mitotic cycles and induces premature endocycles
(Fig. 2A-E). Lack of string at stage 4-6 shows
similar phenotypes (Deng et al., 2001; Schaeffer
et al., 2004). We therefore analyzed whether
overproduction of Hec1/CdhFzr caused a
premature repression of String levels. However, Fig. 5. Ago is dispensable for mitosis but is required for endocycles in follicle cell
no effect on string (or dacapo) expression was epithelium. (A) The ago mRNA is expressed in both mitotic and endocycling follicle
observed in cells that prematurely expressed fzr cells (arrows). (B) Ago is responsible for CycE degradation; follicle cells in ago
and entered endocycles (Fig. 6C,D). (hsFlp;; ago FRT80B/Ubi-GFP FRT80B) clones show increased level of CycE
protein at all stages during oogenesis. Ago loss-of-function does not affect the
Based on these findings, no obvious feedback mitotic cycles in follicle cells: ago clones showed normal expression pattern of stg
regulation was observed between these cell cycle (C; expression prior to st. 6, red arrow, but not past st. 6, black arrow) and the ratio
regulators, disturbed CycE levels did not between the number of cells in sister versus mutant clone was 1:0.9 (D). However,
block endocycles by repressing Hec1/CdhFzr ago clones halt the transition to endocycles: prolonged CycB expression (E,E′),
expression levels and premature Fzr does not small nuclei size (F,F′; clones marked with elevation of CycE levels) and highly
block mitosis by affecting String levels. reduced BrdU incorporation during both endocycles (G,G′) and amplification (H,H′)
Therefore, these data suggest that the Notch- are observed in ago clones. Green, GFP (B,C,E), BrdU (G,H); red, CycE (B, CycE
dependent regulation of these targets is level marks the clones in F-H), β-gal (C), CycB (E); blue, DAPI.
independent of each other (Fig. 7A).

prolong mitotic cycles in posterior follicle cells (Schaeffer et

Discussion al., 2004). This suggests that other critical factors are also
To unravel how the Notch pathway allows follicle cells to regulated in the transition. Interestingly, the regulator of APC-
progress through the mitotic-to-endocycle transition we ubiquitination complex, Hec1/CdhFzr, and the G1 cyclin CycE
previously identified Cdc25 Phosphatase/String as a inhibitor Dacapo are also regulated at the mitotic-to-endocycle
transcriptional responder to Notch activation. However, transition. Hec1/CdhFzr shows transcriptional activation,
overexpression of string is not sufficient to induce the ectopic whereas Dacapo is reduced at the transcriptional level.
mitosis in all follicle cells past stage 6; it can only partly We show that the Notch-dependent cell cycle regulator
3178 Development 131 (13) Research article

Hec1/CdhFzr, that is required for endocycles, is sufficient to bypass the G1-S transition by downregulating the critical
block mitosis and initiate precocious endocycling when checkpoint component p21/Dacapo. However, we have shown
expressed prematurely. However, although the efficiency of that CycE oscillation remains critical for endocycling;
this process is 60%, only 20% of the cells enter premature continuous high level of CycE expression blocks the cell cycle
endocycles (Fig. 2C), which suggests that other components in in G2. The regulation of CycE levels is achieved by the
the G1-S transition might play a role. We also show that a function of F-box/WD40-domain protein Ago/hCdc4 that
critical factor in the mitotic-to-endocycle transition, Cyclin E presumably binds to auto-phosphorylated CycE and directs it
level, is controlled by the posttranslational regulators: the to SCF-complex degradation: high levels of CycE and no
WD40-protein, Ago, and the Notch-responsive CDKI, Dacapo. endocycling is observed in ago-clones. Interestingly, the
Lack of Ago activity or ectopic expression of dacapo lead to function of another G1 cyclin, CycD in growth regulation
a halt in cell cycle progression in the transition. Of these might be compensated by Myc in endocycles, because we have
two CycE regulators, only Dacapo is transcriptionally shown that Myc can affect the kinetics of endocycles. These
downregulated at the transition by activation of the Notch data show that Notch activity executes the mitotic-to-endocycle
pathway. Therefore, Notch allows the follicle epithelial cells to transition by regulating independently all three mitotic
regulators: downregulating the G2
phosphatase Cdc25/String and the G1 CKI
p21CIP/Dacapo and upregulating the
APC activator Hec1/CdhFzr (Fig. 7B).
Repression of String blocks the M-phase,
activation of Fzr allows G1 progression and
downregulation of Dacapo assures entry
into S-phase (Fig. 7A).

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
Fig. 6. The Notch pathway regulates Fzr independently from String and Dacapo. progression (Jacobs et al., 2002; Sigrist and
(A) Although cells ectopicly expressing dacapo (arrowhead) do not undergo endocycles Lehner, 1997). Fzr is required for cyclin
and have smaller nuclei (arrowhead in A′′), they express normal levels of Fzr (arrowhead removal during G1 when the embryonic
in A′′′) compared with wild-type cells (arrow) (hsFlp/fzrG0326; UAS-dap; UASGFP epidermal cell or follicle epithelial
act<FRT-CD2-FRT<Gal4). (B) The normal Fzr expression is observed in most cells proliferation stops and the cells enter
overexpressing dacapo (hsFlp/fzrG0326; UAS-dap; UASGFP act<FRT-CD2-FRT<Gal4) endocycles (Schaeffer et al., 2004; Sigrist
or cyclinE (hsFlp/fzrG0326; UAS-cycE/UASGFP act<FRT-CD2-FRT<Gal4). and Lehner, 1997).
(C) Premature expression of fzr (arrowhead in C′) causes precocious endocycles (large
nuclei, arrowhead in C′′) but does not affect string expression (C′′′, arrowhead indicates
We now show that premature
UAS-fzr cells, arrow – wild-type cells). (D) No effect on string or dacapo expression was Hec1/CdhFzr transcription in follicle cells is
observed upon premature expression of fzr (the diagram shows that UAS-fzr and wild-type sufficient to block mitosis and initiate
follicle cells show close to equal percentage of cells with Dacapo and String staining, precocious endocycling. This suggests that
hsFlp; UAS-fzr/UASGFP act<FRT-CD2-FRT<Gal4;6.4-string-lacZ or dap5gm). Green, Fzr is a powerful player in the mitotic-to-
GFP; red, β-gal; blue, DAPI. endocycle switch, yet regulation of other
 Notch regulates cell cycle transitions 3179

A Mitotic cell cycle Endocycle in follicle cells, suggesting that myc mutant follicle cells
can make the transition from mitosis to the endocycle,
M M but that they can only very inefficiently support the
endocycle (Maines et al., 2004). Therefore, both loss-of-
Notch function and overexpression experiments suggest that Myc is
Notch
G1 G2 pathway G1 an essential component for the proper rate of endocycles in
G2 follicle cells.

S Cyclin E in endocycles
S
In addition to Myc and Cyclin D, Cyclin E also plays an
important role in the regulation of the G1/S-transition. Cyclin
B E binds to and activates the cyclin-dependent kinase Cdk2, and
Notch thereby promotes the transition from G1 to S (Knoblich et al.,
pathway
1994). Oscillation of Cyclin E activity is a mechanism
Cdh/Fzr responsible for the timely inactivation of this G1 cyclin/Cdk
CycA complex and an arrest in cell proliferation. The oscillation of
APC/C
CycB Cyclin E level is controlled partly by a SCF-ubiquitin-
Stg M
dependent proteolysis (Koepp et al., 2001; Moberg et al., 2001;
CycB/Cdk Strohmaier et al., 2001; Won and Reed, 1996). Fluctuations of
Cyclin E are critical for multiple rounds of endocycles (Follette
G2 G1 Myc
et al., 1998; Weiss et al., 1998).
Cyclin E is critical for endocycles in follicle cells as well,
CKI/Dap and our analysis shows that the CycE level is controlled by an
S SCF-regulator, F-box protein, Ago/hCdc4/Fbw7. Fbw7 (Ago)
CycE/Cdk
SCF associates specifically with phosphorylated Cyclin E, and
Ago catalyzes Cyclin E ubiquitination in vitro (Koepp et al., 2001).
Depletion of Ago leads to accumulation and stabilization of
Fig. 7. The mitotic-to-endocycle transition in Drosophila follicle Cyclin E in vivo in human and D. melanogaster. This leads to
cells is executed by Notch-dependent control of cell cycle regulators. increased mitosis in certain mammalian and Drosophila cell
(A) A proposed scheme showing that the Notch pathway induces a types. In addition, ago loss-of-function clones in the germ line
switch from the mitotic-to-endocycle by controlling the activity of will cause extra mitotic divisions or, in contrast, cell cycle
key cell-cycle regulators; cyclin-dependent kinases that control M- arrest and polyploidy (Doronkin et al., 2003). However, we
and S-phase entry and the APC/ubiquitination complex that regulates
degradation of cyclins. Notch activity controls these regulators by
have shown that increased Cyclin E levels observed in ago loss-
downregulating String and Dacapo, and activating Hec1/CdhFzr. of-function mutant clones do not affect the mitotic cycles in
(B) The molecular model in which Notch activity executes the follicle cells but do halt the transition to endocycles that
mitotic-to-endocycle switch by regulating all three major cell cycle normally occurs at stage 6.
transitions. Repression of String blocks M-phase, activation of Why is the function of Ago/hCdc4/Fbw7 critical to
Hec1/CdhFzr allows G1 progression and repression of Dacapo endocycles but not to mitotic cycles in follicle epithelial cells?
assures entry into S-phase. 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.
components is also required for the efficiency of this process. We have shown that dacapo is downregulated at mitotic-to-
Regulators of G1-S transition, such as Dacapo/CIP/KIP, which endocycle transition because of Notch activation and ectopic
also turns out to be a Notch-regulated component, possibly expression of dacapo represses endocycle progression. It is
abort premature attempts by follicle cells to enter the plausible that during mitotic phases Ago and Dacapo share a
endocycle. redundant role for regulating the Cyclin E activity level,
however, dacapo is downregulated by Notch pathway at the
CycD or Myc in growth control time of mitotic-to-endocycle transition and at that point Ago
Our data suggest that a component regulating growth and gains the critical role of sole regulator of Cyclin E protein
thereby the kinetics of G1/S transition in follicle cell activity level. However, downregulation of Dacapo does not
endocycles is the Myc oncogene instead and independent of readily explain the reduction of CycE levels observed in
CycD. In mammals c-Myc controls the decision to divide or mitotic-to-endocycle transition (Fig. 3C). We detected
not to divide and thereby functions as a crucial mediator of elevation of CycE protein level in response to Dacapo
signals that determine organ and body size (Levens, 2003; overexpression, pointing out that this CKI may stabilize CycE
Trumpp et al., 2001). Interestingly, overexpression of dmyc in an inactive form. One possibility therefore is that less CycE
in follicle cells does not affect the mitotic cycles but induces, protein is observed after the Dacapo downregulation because
instead, extra endocycles. Because the timing for entering and Dacapo is no longer stabilizing it.
exit from the endocycles has not changed, however, increased Why is Dacapo downregulated at the time of endocycle
ploidy is observed, we suggest that the rate of endocycles is transition? Expression of Dacapo is important for proper cell
increased because of the overexpression of Myc. This finding cycle regulation. For example, during vertebrate development,
is in accordance with recent loss-of-function analysis on myc members of the CIP/KIP family of CKIs are often upregulated
3180 Development 131 (13) Research article

as cells exit the mitotic cycle and begin to terminally Heart Association, American Cancer Society and Pew Memorial Trust
differentiate. Also, reduced expression of p27Kip1 was and Pew Scholarship in the Biomedical Sciences to H.R.-B.
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 References
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(Mantel et al., 1999). In the Drosophila germ line Dap is Drosophila. Mech. Dev. 110, 71-83.
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High Dap levels in the oocyte are critical to the maintenance Drosophila: a model for metazoan origins of DNA replication and S-phase
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of Dap drive the endocycle (Hong et al., 2003). In contrast to Calvi, B. R., Lilly, M. A. and Spradling, A. C. (1998). Cell cycle control of
all these examples, in endocycling follicle cells reduction chorion gene amplification. Genes Dev. 12, 734-744.
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