Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA
* Author for correspondence (e-mail: mlilly{at}helix.nih.gov)
Accepted 20 December 2002
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SUMMARY |
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Key words: Oogenesis, Meiosis, dacapo, p27, Drosophila, Cyclin E, Oocyte differentiation
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INTRODUCTION |
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Ovarian cyst formation begins when one, out of a group of two to three
germline stem cells, divides asymmetrically to produce a cystoblast. The
cystoblast undergoes a series of four synchronous mitotic divisions in which
cytokinesis is incomplete (de Cuevas et
al., 1997; McKearin,
1997
). Individual cells within the cyst, referred to as
cystocytes, are connected in an invariant pattern by actin-rich intercellular
bridges called ring canals (Fig.
1A). After all 16 cystocytes complete premeiotic S phase, a
meiotic gradient develops with the two cells with four ring canals having the
most meiotic features as assayed by the presence of mature synaptonemal
complexes and recombination nodules
(Chandley, 1966
;
Carpenter, 1975
;
Carpenter, 1981
;
Schmekel et al., 1993
).
Ultimately, the cell cycles of the future nurse cells and oocyte diverge
dramatically. The single pro-oocyte arrests in prophase of meiosis I for most
of the growth phase of oogenesis, while the 15 nurse cells go on to complete
10-12 endocycles to become highly polyploid. During these endocycles the nurse
cells cycle asynchronously.
|
How one of the 16 cystocytes is selected to differentiate as the oocyte has
long been an issue of interest. It has been known for some time that one of
the two cells with four ring canals always develops as the oocyte (reviewed by
Büning, 1994). This
observation indicates that the invariant pattern of cystocyte connections is
crucial for later cell fate decisions within the cyst (reviewed by
de Cuevas et al., 1997
;
McKearin, 1997
). Studies over
the last few years suggest that oocyte differentiation is a two-step process.
The first step entails the establishment of an asymmetry between the
pro-oocyte and the pro-nurse cells. Exactly when and how this asymmetry is
established is unknown, although it may occur as early as the cystoblast
division through the unequal distribution of the fusome, a germline-specific
organelle (Lin and Spradling,
1995
; de Cuevas and Spradling,
1998
). The second step involves the directional transport of
cellular components, including specific mRNAs and proteins, to the pro-oocyte.
Drugs that destabilize microtubules eliminate the accumulation of specific
mRNAs in the pro-oocyte and lead to the production of cysts that contain 16
nurse cells and no oocyte (Koch and
Spitzer, 1985
; Theurkauf et
al., 1993
). Similarly, recessive mutations in the genes
Bicaudal D (BicD) and egalitarian (egl)
prevent the differential accumulation of specific mRNAs and proteins in the
pro-oocyte and result in cysts with 16 nurse cells
(Suter et al., 1989
;
Schupbach and Wieschaus, 1991
;
Suter and Steward, 1991
;
Mach and Lehmann, 1997
).
Although it is clear that microtubule-based polarized transport is a critical
component of oocyte differentiation, exactly why meiosis proceeds in the
oocyte while the adjacent nurse cells enter the endocycle remains
undefined.
During meiosis, the integrity of the genome must be maintained through
recombination and two rounds of cell division. Animals in which the oocyte
develops within the context of a cyst, such as Drosophila, are faced
with an additional set of challenges. Intercellular bridges physically connect
the Drosophila oocyte to nurse cells that are undergoing repeated
rounds of DNA replication. However, to produce a functional gamete, it is
essential that the oocyte does not fire a single DNA replication origin. How
does the germline cyst maintain this extreme cell cycle dichotomy? One
potential mechanism involves the spatial regulation of cyclinE-Cdk2 activity
(Lilly and Spradling, 1996).
In Drosophila, cyclinE-Cdk2 activity is required for S phase
(Knoblich et al., 1994
).
dacapo (dap) is a vital gene that encodes a
p21CIP/p27KIP1/p57KIP2-like cki that
specifically inhibits the activity of cyclinE-Cdk2 complexes
(de Nooij et al., 1996
;
Lane et al., 1996
). Throughout
much of the growth phase of Drosophila oogenesis, the levels of the
cki Dap oscillate in the 15-polyploid nurse cells but remain persistently high
in the single oocyte (de Nooij et al.,
2000
). We reasoned that the differential regulation of Dap might
provide a mechanism to maintain the oocyte in prophase of meiosis I, while
allowing the endocycle to proceed in the adjacent nurse cells.
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MATERIALS AND METHODS |
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Generation of dap4 germline clones
Germline clones were generated using the FLP:FRT system
(Chou and Perrimon, 1996).
Specifically, y, w, FLP12; FRT42B arm-lacZ females were crossed to
w; FRT42B, dap4/CyO males. Eggs produced from
this cross were heat-shocked for 1 hour at 37°C in a circulating water
bath 96 hours after egg deposition. A second identical heat shock was
administered 24 hours later. After eclosion y, w, FLP12; FRT 42B
arm-LacZ/FRT42B, dap4 adult females were collected and
heat-shocked at least once for 1 hour at 37°C. Females were allowed to
recover for 8 days at 25°C. Clones were negatively marked by the absence
of ß-galactosidase protein as determined by immunocytochemistry.
Immunohistochemistry and BrdU labeling
Ovaries were dissected, immunologically stained and mounted as described
previously (Lin et al., 1992).
Dap monoclonal antibody (de Nooij et al.,
2000
) was provided by Iswar Hariharan. Dap rabbit polyclonal
antibody was provided by Christian Lehner. Orb monoclonal antibody
(Lantz et al., 1994
) was
provided by the Developmental Studies Hybridoma Bank. BicD monoclonal antibody
(Junyoung and Steward, 2001
)
was provided by Ruth Steward. Ovaries were fixed in methanol/EGTA for tubulin
staining.
-Tubulin monoclonal antibody DM1a (Sigma) was used at 1:500.
BrdU staining was essentially as described by Avedisov et al.
(Avedisov et al., 2000
). C(3)G
antibody was generated from 301 amino acid C-terminal fragment in rabbits
using standard techniques. The C(3)G antibody was used at 1:3000 dilution.
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RESULTS |
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The differential behavior of Dap in the nurse cells versus the oocyte is
first observed in late stage 1 egg chambers. Although high levels of Dap
persist in the oocyte, the levels of Dap begin to oscillate in the nurse cells
as they asynchronously enter the endocycle
(Fig. 2C,D)
(de Nooij et al., 2000). Dap
levels continue to oscillate in the nurse cells until stage 10 of oogenesis
when the nurse cells stop replicating their DNA
(Fig. 2F). By contrast, Dap
levels remain high in the oocyte until well after the nurse cells exit the
endocycle. Dap inhibits DNA replication in both mitotic and endocycling cells
(de Nooij et al., 1996
;
Lane et al., 1996
;
Calvi et al., 1998
). Thus, it
is likely that the cycling of Dap protein in the nurse cells allows
cyclinE-Cdk2 kinase activity to rise high enough to trigger the endocycle S
phases (de Nooij et al., 2000
;
Edgar and Orr-Weaver, 2001
).
Consistent with this proposal, S phase in the nurse cells occurs when Dap
levels are low (Fig. 2G,H).
Therefore, as the nurse cells enter the endocycle in stage 1 there is a clear
correlation between the differential regulation of Dap and the distinct cell
cycles of the nurse cells and oocyte.
dap oocytes enter the endocycle
Does the cki Dap prevent the oocyte from entering the endocycle with the
nurse cells and thus preserve the prophase I meiotic arrest? To answer this
question, we generated homozygous germline clones of the dap null
allele dap4 (Lane et
al., 1996). dap4 contains a deletion of the
conserved CDK-binding domain and acts as a complete loss-of-function allele
(Lane et al., 1996
). In
greater than 80% of the egg chambers (n>250) that contain
dap germline clones, the oocyte enters the endocycle and becomes
polyploid (Fig. 3). The extent
of polyploidy in these oocytes is variable. 53±9% of dap cysts
have 16-polyploid nurse cells and no oocyte
(Fig. 3B). In this phenotypic
class, all cells in the cyst have similar DNA contents, indicating all 16
cells have undergone approximately the same number of endocycles. These data
suggest that in greater than 50% of the egg chambers with dap
germline clones, the oocyte enters the endocycle at the same time as the nurse
cells. In 28±7% of the germline clones the oocyte is polyploid, but can
be distinguished from the adjacent nurse cells by its lower DNA content and
posterior position (Fig. 3C).
In wild-type egg chambers the oocyte DNA condenses into a compact karyosome in
stage 3 of oogenesis (Fig. 3A). In the 10±6% of dap clones in which the oocyte is not
obviously polyploid, karyosome formation is often aberrant. For example, the
oocyte DNA is present in one or more elongated masses within the oocyte
nucleus (Fig. 3D) or in a thin
rim near the nuclear envelope (data not shown). The
9% of dap
clones that contained greater than 16 nurse cells were not included in this
analysis. These data indicate that dap regulates entry into or is
required for the maintenance of the meiotic cycle in the oocyte.
|
What accounts for the observed variability of the dap phenotype? Because we examined the clonal progeny from germline stem cell clones that had undergone numerous divisions, we do not believe the observed phenotypic variability results from the perdurance of the Dap protein. In support of this conclusion, immunocytochemistry using an antibody against Dap indicated that clonal mutant egg chambers contain no Dap protein (data not shown). In addition, a similar phenotypic distribution was observed when examining the ovaries from dap4 homozygous escapers. Finally, dap germline cysts surrounded by dap4 (n=88 egg chambers) and dap+ (n=104 egg chambers) follicle cells had similarly variable phenotypes, indicating that the genotype of the follicle cells is not the source of variability. Protecting the oocyte from inappropriate entry into the endocycle is critical to the production of a functional gamete. Therefore, we predict that additional factors act in concert with Dap to inhibit DNA replication during meiosis.
dap is not required for oocyte specification or entry into
meiosis
Mutations that disrupt cyst polarity and oocyte specification, such as
BicD and egl, result in the failure to localize oocyte
specific markers and the production of egg chambers with 16 nurse cells and no
oocyte (Suter et al., 1989;
Suter and Steward, 1991
;
Christerson and McKearin, 1994
;
Lantz et al., 1994
). We wanted
to determine if mutations in dap indirectly influence the oocyte cell
cycle by disrupting cyst polarity. In wild-type cysts, the Orb and BicD
proteins preferentially accumulate in the cytoplasm of the pro-oocyte,
beginning in region 2a of the germarium
(Suter and Steward, 1991
;
Lantz et al., 1994
). With few
exceptions, the distribution of the Orb
(Fig. 4A) and BicD (data not
shown) proteins in dap cysts is indistinguishable from wild type in
region 2 of the germarium (Fig.
4A). As shown in Fig.
4, Orb protein preferentially accumulates in a single centrally
located cystocyte in both wild-type (arrow) and dap (arrowhead)
cysts. These data indicate that unlike what is observed in BicD and
egl mutants, the polyploidization of dap oocytes is not the
consequence of a failure to specify an oocyte or a general disruption in cyst
polarity. However, it should be noted that in egl mutants, Dap
protein does not preferentially accumulate within a single cell in
post-germarial egg chambers, but instead appears to oscillate in all 16-cyst
cells as they undergo repeated endocycles (data not shown). Thus, although
dap is not required for oocyte specification, the specification of
the oocyte is required to establish the two modes of Dap regulation within
ovarian cysts. These data indicate that oocyte specification is upstream of
the effects of Dap on the meiotic cycle.
|
In the absence of Dap the majority of oocytes do not undergo the two
meiotic divisions but instead enter the endocycle and replicate their DNA.
Next, we wanted to examine if Dap is required for the initiation and/or the
maintenance of meiosis? In order to determine if dap oocytes enter
meiosis, we assayed meiotic progression using an antibody directed against the
synaptonemal complex (SC) component C(3)G
(Page and Hawley, 2001). In
wild-type cysts, up to four cells construct SC with the two pro-oocytes
entering pachytene (Carpenter,
1975
; Carpenter,
1979
; Huynh and St Johnston,
2000
; Page and Hawley,
2001
) (Fig. 4B). By
late region 2b, the meiotic gradient sharpens such that C(3)G staining is
primarily concentrated in the oocyte. dap oocytes progress to
pachytene as assayed by the production of continuous SC along bivalents
(Fig. 4C). These data indicate
that dap is not necessary to initiate the meiotic cycle nor is it
necessary for progression to pachytene. Therefore, the first disruption to the
meiotic cycle observed in dap oocytes is when they inappropriately
enter the endocycle in stage 1. These data are consistent with Dap acting to
inhibit DNA replication specifically in the oocyte as the adjacent nurse cells
enter the endocycle.
In dap germline cysts there is a delay in the restriction of SC to
a single cell such that cysts in region 2b and 3 occasionally have 2 or more
cells with strong C(3)G staining (data not shown). We believe it is unlikely
that the retention of SC in the adjacent nurse cells is responsible for the
subsequent polyploidization of the oocyte observed in dap mutants.
The spindle mutants, which activate the meiotic checkpoint
(Ghabrial et al., 1998), show
a very similar delay in the restriction of SC to the oocyte
(Huynh and St Johnston, 2000
).
Importantly, mutations in the spindle genes do not result in the
polyploidization of the oocyte. We believe the delay in the restriction of SC
to a single cell may represent an earlier function of dap, perhaps
during the mitotic cyst divisions or in the regulation of premeiotic S
phase.
dap is required to maintain oocyte differentiation
In stage 1 egg chambers the cell-cycle environment within the cyst changes
dramatically as the nurse cells asynchronously enter the S phase of the first
endocycle. In wild-type stage 1 egg chambers, the oocyte remains safely
arrested in prophase of meiosis I. However, in the majority of dap
mutant cysts, the oocyte enters the endocycle with the nurse cells. Our data
indicate that inappropriate entry into the endocycle disrupts oocyte
differentiation (Fig. 5).
Beginning at stage 1, we observe a decrease, relative to similarly aged
wild-type egg chambers, in the preferential accumulation of the BicD and Orb
proteins in dap oocytes. As oogenesis progresses, there is a strong
inverse correlation between the degree of polyploidization in dap
oocytes and the preferential accumulation of BicD and Orb. dap cysts
with highly polyploid oocytes have little to no preferential accumulation of
BicD (Fig. 5C,D) or Orb (data
not shown). By contrast, dap cysts in which the oocyte has undergone
limited polyploidization frequently have BicD and Orb levels indistinguishable
from wild type (Fig. 5E,F). As
is observed in other mutants that disrupt oocyte differentiation, egg chambers
that contain dap clones rarely develop beyond stage 6
(Suter et al., 1989;
Clark and McKearin, 1996
;
Mach and Lehmann, 1997
;
Huynh et al., 2001
). In
Drosophila vitellogenesis begins in stage 7 when the oocyte begins to
take up large quantities of yolk. The small percentage of dap oocytes
that progress far enough to take up yolk, invariably have undergone little to
no polyploidization (data not shown). These data demonstrate that dap
is required for oocyte differentiation. In addition, they indicate that the
loss of oocyte identity observed in dap clones is a direct
consequence of the oocyte entering the endocycle.
|
To explore further the apparent loss of oocyte identity that accompanies
inappropriate entry into the endocycle, we examined the distribution of
microtubules in dap cysts. The preferential accumulation of Orb and
BicD in the oocyte is dependent on a polarized network of microtubules that
directs these, and other oocyte-specific factors, from the nurse cells to the
oocyte (Theurkauf et al.,
1993). The disruption of this network by microtubule
depolymerizing agents leads to the production of egg chambers with 16
polyploid nurse cells (Koch and Spitzer,
1985
; Theurkauf et al.,
1993
; Huynh and St Johnston,
2000
). In wild-type cysts, the asymmetric distribution of
microtubules within the germline cyst can be visualized as a preferential
accumulation of
-tubulin in the single oocyte, which contains the
microtubule-organizing center (Theurkauf
et al., 1992
). In dap cysts, this focus of
-tubulin staining is present in nonpolyploid oocytes but absent in
polyploid oocytes (Fig. 6).
These data suggest that entry into the endocycle may disrupt the polarized
microtubule network, which in turn blocks oocyte differentiation. However, the
exact relationship between entry into the endocycle and the disruption of the
microtubule network remains undefined.
|
dap dominantly suppresses the two oocyte phenotype of a cycE
hypomorph
In females homozygous for the hypomorphic mutation
cycE01672, a fraction of egg chambers contain two cells
that have oocyte-like nuclear features, such as low ploidy values, an endobody
and a small DNA mass in a very large nucleus
(Lilly and Spradling, 1996).
Egg chambers that contain two oocyte nuclei have only 14 polyploid nurse
cells, indicating that a cell that was destined to develop as a nurse cell has
been partially transformed towards the oocyte fate. The extra oocyte nucleus,
which can be distinguished from the true oocyte by its presence in a small
cell that lacked signs of cytoplasmic oocyte differentiation, almost
invariably is the other four-ring canal cell in the cyst
(Lilly and Spradling, 1996
).
Interestingly, these transformed nuclei accumulate persistently high levels of
Dap protein in a manner similar to the true oocyte
(Fig. 7A,B). Cells with
persistently high levels of Dap have low ploidy values, indicating they have
either not entered the endocycle or have prematurely exited the cycle. By
contrast, in wild-type egg chambers the other four-ring canal cell develops as
a highly polyploid posterior nurse cell in which Dap levels oscillate. Thus,
mutations in dap result in germline cysts in which all 16 cells enter
the endocycle and develop as nurse cells, while a mutation in cycE
has the opposite effect, resulting in two or more cells that have persistently
high levels of Dap that cannot enter and/or maintain the endocycle.
|
To examine the relationship between cycE and dap in the
regulation of the cell-cycle program of ovarian cysts, we examined if
mutations in dap could dominantly modify the
cycE01672 two oocyte phenotype. We found that reducing the
dose of the dap gene by half, resulted in 2.5 fold suppression
of the cycE01672 two oocyte phenotype. In wild-type egg
chambers the four posterior nurse cells, which are connected to the oocyte via
ring canals, have the highest ploidy values in the cyst
(Fig. 7C). In
cycE01672 females 37±7% (n>200) of egg
chambers contain a cell adjacent to the true oocyte with inappropriately low
ploidy values (Fig. 7D,E). When
a single copy of the null allele dap4 was placed in the
cycE01672 background, fewer than 14±7%
(n>200) of egg chambers had a posterior nurse cell with a reduced
DNA content. These data indicate that whether a cyst cell enters and/or
maintains the endocycle is at least partially determined by the balance of
CycE and Dap. In addition, they strongly suggest that, as is observed during
embryogenesis, the primary target of Dap in the ovary is the CycE/Cdk2
complex.
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DISCUSSION |
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A model for the maintenance of the meiotic cycle in
Drosophila oocytes
Our observations suggest a model for how the meiotic cycle and the
endocycle are independently maintained within Drosophila ovarian
cysts (Fig. 8A). We propose
that the presence of high levels of the cki Dap in the oocyte, throughout the
time the nurse cells are in the endocycle, persistently inhibits cyclinE-Cdk2
kinase activity and prevents inappropriate DNA replication during meiosis.
Without the inhibition of cyclinE-Cdk2 kinase activity provided by high levels
of Dap, the majority of dap mutant oocytes abandon the meiotic cycle
and enter the endocycle with the nurse cells. Importantly, these data indicate
that, as has recently been observed in mice, oocytes in prophase of meiosis I
are competent to replicate their DNA
(Czolowska and Borsuk, 2000).
In the mouse oocyte, the inhibition of DNA replication during prophase of
meiosis I may be accomplished through the downregulation of the G1 cyclins and
Cdk2 (Moore et al., 1996
;
Czolowska and Borsuk, 2000
).
In the Drosophila oocyte, the inhibition of cyclinE-Cdk2 activity by
Dap achieves the same aim. In contrast to the oocyte, the nurse cells require
a period when Dap levels are low to allow cyclinE-Cdk2 kinase activity to rise
high enough to trigger each endocycle S phase. These low points occur during
the oscillations of the Dap protein. Our data indicate that it is through the
differential regulation of Dap that two apparently incompatible cell cycles
are stably maintained within the common cytoplasm of the ovarian cyst.
|
The regulatory relationship between cyclinE-Cdk2 activity and the Dap
ortholog p27 suggest a feedback loop that may account for the long-term
stabilization of Dap in post germarial oocytes. In mammalian cells,
phosphorylation by cyclinE-Cdk2 targets the p27 protein for destruction by the
proteasome (Pagano et al.,
1995; Sheaff et al.,
1997
; Vlach et al.,
1997
; Montagnoli et al.,
1999
). Similarly, the Dap protein contains a CDK phosphorylation
consensus site (Ser205) and can be phosphorylated by mammalian
cyclinE-Cdk2 in vitro (de Nooij et al.,
1996
). We propose that in early stage 1 egg chambers, the balance
of cyclinE-Cdk2 activity and Dap protein is slightly different in the 15-nurse
cells versus the single oocyte (Fig.
8B). In the oocyte, the balance is tipped towards the inhibitor
Dap, resulting in diminished cyclinE-Cdk2 activity. Lower cyclinE-Cdk2
activity leads to a reduced rate of Dap phosphorylation and proteolysis,
thereby increasing the concentration of the Dap protein. The stabilization of
the Dap protein ultimately results in the permanent inhibition of cyclinE-Cdk2
activity in the oocyte. In contrast to the oocyte, in stage 1 nurse cells
cyclinE-Cdk2 kinase activity reaches high enough levels to trigger the
phosphorylation and subsequent destruction of the Dap protein, thus allowing
endocycle progression. The above model predicts that additional proteins that
are targeted for destruction by cyclinE-Cdk2 phosphorylation should be
stabilized in the oocyte but not in the nurse cells. Like p27, the proteolytic
destruction of CycE itself is dependent on phosphorylation by the cyclinE-Cdk2
complex (Clurman et al., 1996
;
Won and Reed, 1996
). As
predicted by the model, CycE is stabilized in the oocyte and accumulates to
high levels as oogenesis progresses (Lilly
and Spradling, 1996
). This model allows a slight difference in the
balance of cyclinE-Cdk2 activity and Dap early in oogenesis to be amplified,
resulting in the two cell types of the germline cyst permanently adopting
dramatically different cell cycles.
Consistent with the above model, removing one copy of dap dominantly inhibits the two oocyte phenotype observed in the cycE01672 hypomorph. In cycE01672 females, Dap protein frequently accumulates to high levels in one or more cyst nuclei in addition to the true oocyte. The cells that inappropriately stabilize Dap have low DNA contents and are connected to the true oocyte through a ring canal. In effect, it appears that in the cycE01672 mutants, the stabilization of Dap and the accompanying inhibition of the endocycle that is normally restricted to the oocyte is allowed to spread outwards to the adjacent nurse cells. Thus, reducing CycE levels favors the stabilization of Dap, which leads to the inappropriate inhibition of the endocycle in cells connected to the true oocyte. Importantly, we demonstrate that this phenotype is suppressed by removing a single copy of the dap gene from the cycE01672 background. Thus, by bringing CycE and Dap levels back into balance, the nurse cells near the oocyte are no longer inappropriately drawn into the feedback loop that inhibits the endocycle.
However, an important question remains. Beyond the specification of the
oocyte, what molecular pathway accounts for the proposed difference in the
balance of cyclinE-Cdk2 activity and Dap in nurse cells relative to the oocyte
in early stage 1 egg chambers? There are at least three possible mechanisms,
not mutually exclusive, that may explain this initial asymmetry. Intriguingly,
dap mRNA is transported to the oocyte beginning in region 2 of the
germarium and remains at high levels in the oocyte throughout oogenesis
(de Nooij et al., 2000). Thus,
the oocyte may simply be able to translate more Dap protein and therefore keep
Dap levels slightly higher in the oocyte. This slight difference may be below
the resolution of immunocytochemistry, thus explaining why Dap appears to be
evenly distributed in early stage 1 egg chambers. Alternatively, cyclinE-Cdk2
activity may be partially inhibited in stage 1 oocytes via a Dap-independent
mechanism. This possibility is supported by the observation that even in the
complete absence of Dap, not all oocytes enter the endocycle, indicating that
oocytes have an alternative mechanism to inhibit inappropriate DNA
replication. Finally, Dap proteolysis may be less efficient in the oocyte for
reasons independent of the level of cyclinE-Cdk2 activity, such as a general
inhibition of the proteasome. The identification of additional genes that
influence the maintenance of the meiotic cycle during oogenesis will help
distinguish between the above possibilities.
Cell-cycle regulation and cellular differentiation in ovarian
germline cysts
Studies over the last few years indicate that the differentiation of both
the nurse cells and oocyte are strongly influenced by cell-cycle events within
the germline cyst (Lilly and Spradling,
1996; Ghabrial et al.,
1998
; Ghabrial and Schupbach,
1999
; Myster et al.,
2000
; Page et al.,
2000
). Our data support the conclusion that cell-cycle regulation
and oocyte differentiation are closely coupled. Specifically, we find that
inappropriate entry into the endocycle disrupts oocyte differentiation in
dap germline cysts and results in the presumptive oocyte developing
like a nurse cell. The more polyploid the oocyte, the greater the disruption
in oocyte differentiation as measured by the oocyte-specific accumulation of
the proteins BicD and Orb, as well as yolk uptake. In mutants like
dap, where the oocyte is specified but ultimately enters the
endocycle, cause and effect can be difficult to determine. Does the oocyte
enter the endocycle because of the inability to maintain the oocyte fate or
alternatively does entry into the endocycle disrupt the ability to maintain
the oocyte identity? Considering the known role of dap in cell-cycle
regulation, we believe that the phenotype observed in dap mutants
reflects the second scenario. Our data indicate that entry into the endocycle
is incompatible with many aspects of the oocyte developmental program and can
serve as the primary cause of the loss of oocyte identity. The identification
of additional genes that influence the maintenance of both the nurse cell and
oocyte identities will help clarify the exact role of cell-cycle programming
in the nurse cell/oocyte fate decision of Drosophila ovarian
cysts.
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ACKNOWLEDGMENTS |
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