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First published online 13 September 2006
doi: 10.1242/dev.02565
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1 Inserm, U384, Clermont-Ferrand, F-63001 France; Univ Clermont 1, UFR
Médecine, 28, place Henri Dunant, Clermont-Ferrand, F-63001
France.
2 Hybrigenics SA, 3-5 Impasse Reille, 75014 Paris, France.
* Author for correspondence (e-mail: jl.couderc{at}inserm.u-clermont1.fr)
Accepted 3 August 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Polo, BicD, Oocyte, Meiosis, Polarized transport, Drosophila
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Riechmann and Ephrussi,
2001).
Drosophila oogenesis begins in a structure called the germarium,
which is divided into several regions (Fig.
2A) (Spradling,
1993). In its anterior part, named region 1, germline stem cell
progeny undergoes a precise pattern of divisions to form cysts of 16 cells
interconnected by cytoplasmic bridges, the ring canals. Oocyte differentiation
is a progressive process that begins in region 2a by the selection of two
pro-oocytes corresponding to the first two cells of the cyst. As the cyst
enters region 2b and contacts the follicle cells, one cell is selected to
become the oocyte, and the other 15 cells will differentiate into nurse cells.
This progressive specification can be observed by the accumulation of mRNA and
proteins such as Bicaudal D (BicD), and by the migration of the centrioles
(Fig. 2A)
(Bolivar et al., 2001;
Cox and Spradling, 2003;
Ephrussi et al., 1991;
Keyes and Spradling, 1997;
Suter et al., 1989;
van Eeden et al., 2001). When
the cyst progresses from region 2b to region 3, it starts to round up, with
the oocyte always positioned at the posterior. At this step, centrosomes,
mRNAs, proteins and organelles found at the anterior of the oocyte move to the
posterior. This early polarization event is important as it prefigures the
future antero-posterior axis of the embryo
(Huynh et al., 2001).
In each cyst, a germline-specific membranous structure called the fusome
extends asymmetrically throughout the ring canals in all 16 cells
(Fig. 2A)
(de Cuevas et al., 1996;
Lin et al., 1994). This
asymmetric distribution is thought to determine which cell becomes the oocyte
(de Cuevas and Spradling, 1998;
Lin and Spradling, 1995;
Lin et al., 1994;
Yue and Spradling, 1992).
However, this initial asymmetry alone is not sufficient to understand oocyte
differentiation, and genetic analyses are required to distinguish different
steps in the differentiation process.
The polarization of the germline cyst relies on microtubule-dependent
transport processes. Microtubules and dynein are required for the accumulation
of oocyte determinants, such as BicD protein, and thus for oocyte
differentiation (Theurkauf et al.,
1993; Bolivar et al.,
2001). The transport of mRNA and proteins to the oocyte is also
dependent on BicD and Egl proteins (Bolivar
et al., 2001; Clark and
McKearin, 1996; Navarro et
al., 2004; Ran et al.,
1994; Schupbach and Wieschaus,
1991; Suter et al.,
1989). These proteins interact with each other, and both are able
to interact with different subunits of the Dynein complex
(Hoogenraad et al., 2001;
Mach and Lehmann, 1997;
Navarro et al., 2004). BicD
may function as an adaptor for cargo molecules such as mRNA, and it has been
suggested that Egl is an important regulator of this function. Finally, the
early polarization of the oocyte in region 2b-3 involves many genes including
the dynein light chain 8 and, again, egl and BicD
(Huynh and St Johnston, 2000;
Navarro et al., 2004).
All these functional steps are required for establishing or maintaining oocyte fate. Each mutation that disrupts this process leads to the formation of cysts that have neither an oocyte nor a cell in meiosis, and instead consist of 16 endoreplicative nurse cells. Thus, meiosis control is dependent on oocyte determination.
During Drosophila oogenesis, meiosis starts with homologous
recombination that can be recognized through the formation of the synaptonemal
complexes (SCs) and the recruitment of proteins such as C(3)G
(Huynh and St Johnston, 2000;
Page and Hawley, 2001;
Hong et al., 2003;
Carpenter, 1975). Meiosis
begins in region 2a of the germarium, usually in four cells of a cyst
(Fig. 2A). Meiosis is quickly
restricted to the two pro-oocytes, then to the oocyte as the cyst progresses
into region 2b. Therefore, meiotic control appears to be spatially and
temporally correlated with oocyte determination, and it is difficult to
determine whether one process precedes the other.
Functional studies have provided further evidence on the links between
oocyte determination and meiosis. Null mutations of egl and
BicD have been described to have dramatic and opposite effects on
meiosis (Huynh and St Johnston,
2000). In BicD mutant cysts, no cells possess SCs,
whereas all the cells of egl mutant cysts form SCs in region 2a
before all of them exit meiosis simultaneously. Although the initial
difference between these two mutants is not yet understood, this observation
shows that both are involved in the initial restriction of meiosis to four
cells. Finally, proteins required for early oocyte polarization are also
required for maintaining the oocyte in meiosis after its restriction to one
cell (reviewed by Huynh and St Johnston,
2004). Therefore, apart from the essential role of BicD and Egl,
the spatiotemporal control of meiosis remains poorly understood.
Obviously, initiation of meiosis is itself under the control of classical
cell-cycle regulators. Partial loss-of-function mutations in cyclin E, the
main cyclin controlling replication and endoreplication, can lead to the
formation of 16-cell cysts containing two meiotic cells, both presenting
oocyte-like nuclear and cytoplasmic features
(Lilly and Spradling, 1996).
Conversely, a mutation in p27cip/dacapo, a negative regulator of
cyclin E, induces the formation of cysts with 16 endoreplicative nurse cells
and no oocyte (Hong et al.,
2003). These findings allow the following conclusions. First,
meiosis and endoreplication seem to act in competition, as the reduction of a
positive or negative determinant of one process promotes or represses the
other, respectively. Second, the cell cycle decision of a cell is sufficient
to determine its fate, as both oocyte and nurse cells can be led to adopt the
other fate by altering the control of the cell cycle. Finally, these results
strongly suggest that the choice between endoreplication and meiosis involves
the asymmetric distribution of cell-cycle regulators, and this asymmetry may
depend on the general process of cyst polarization. One candidate for an
asymmetric meiotic determinant is Dacapo, as it is found specifically in the
oocyte nucleus in region 3 of the germarium. However, this asymmetric
distribution is not observed at earlier stages, and a null mutant for
dacapo does not affect meiotic progression in region 2 but only its
maintenance in region 3. Many other proteins involved in cell-cycle control
have been implicated in oocyte specification, thus confirming their influence
on cell fate decisions. However, to date, no cell-cycle regulator has been
found to be asymmetrically localized early enough to explain how the balance
between meiosis and endoreplication is initially controlled. Moreover, how
cell-cycle control influences oocyte cell fate decision remains unknown.
In this article, we show that the Polo kinase, one of the main regulators of the G2/M transition, interacts with BicD protein during oogenesis. Genetic analyses reveal interdependent functions between both proteins during early meiosis control and oocyte specification. As Polo plays a role in cell-cycle control and BicD plays a role in polarized transport to the future oocyte, we propose that their interaction reflects the existing link between meiosis and oocyte determination.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Details can be
provided upon request.
Drosophila strains and genetics
All the crosses were produced at 25°C using standard manipulation of
fly genetics. Transgenic lines of UASp-polo construct were generated by
standard methods and two independent lines were analyzed. Clonal analysis was
performed with the FLP/FRT system (Xu and
Rubin, 1993) using nuclear GFP as a clone marker.
Yeast two-hybrid
The yeast two-hybrid screens were performed with Plk1 fragments as baits to
screen a human placenta cDNA library using a previously described mating
method (Formstecher et al.,
2005).
Immunostaining
Tissue stainings were performed according to standard procedures, using the
primary antibodies at the following dilutions: rabbit anti-C(3)G antibody at
1/1000 (Hong et al., 2003;
Lilly and Spradling, 1996);
rabbit CP309 antibody 1/500 (Kawaguchi and
Zheng, 2004); mouse anti-Polo MA294 1/10
(Llamazares et al., 1991);
mouse anti-Hts 1B1 1/100 (Developmental Studies Hybridoma Bank); mouse
anti-BicD 1B11 plus 4C2 at 1/50 each (Developmental Studies Hybridoma Bank).
Cy3, Cy5 (Jackson Immunoresearch) and Alexa 488-conjugated secondary
antibodies (Molecular Probes) were used at 1/500.
Ovary immunoprecipitation
Immunoprecipitation was performed as described in Navarro et al.
(Navarro et al., 2004) using
polyclonal anti-GFP antibody (Clontech). Details can be provided upon
request.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Elia et al., 2003). Deletions
at both extremities show that the entire Polo-Box domain is both necessary and
sufficient for the two-hybrid interaction with BicD
(Fig. 1A).
To date, the only link identified between BicD and cell cycle concerns
entry into meiosis during Drosophila oogenesis
(Huynh and St Johnston, 2000).
Moreover, the interaction domain of BicD with Polo is particularly well
conserved and has been shown to be functionally significant, especially during
early oogenesis (Oh et al.,
2000). Therefore, we tested the ability of both proteins to
interact using co-immunoprecipitation on ovary extracts. We took advantage of
flies containing a GFP-Polo transgene that has been shown to reproduce Polo
expression and localization in all cell types analyzed, and to rescue
polo mutants (Moutinho-Santos et
al., 1999). Wild-type flies and flies constitutively expressing an
NlsGFP protein were used as negative controls. Anti-GFP antibody efficiently
precipitates both NlsGFP and GFP-Polo proteins but BicD was coprecipitated
only with the GFP-Polo (Fig.
1B). This experiment shows that Polo and BicD proteins interact in
vivo during Drosophila oogenesis.
Polo is gradually restricted to meiotic cells during cyst polarization
We analyzed Polo localization during oogenesis using flies that were
hemizygous for the GFP-Polo construct. In the germarium, Polo was strongly
expressed in all the germline cells of region 1, suggesting that the presence
of Polo is not cell-cycle-dependent (Fig.
2B). At the subcellular level, GFP-Polo accumulated in several
cytoplasmic dots in each cell, generally at the nuclear periphery in region 1
(Fig. 2C). In region 2a, the
dots became progressively less bright, except in the more central part of the
cysts where they remained particularly intense
(Fig. 2D). In regions 2b and 3,
Polo was found in one or sometimes a few prominent dots at the posterior of
the cyst (Fig. 2B,E). This
localization was maintained until stages 2-3, and then became undetectable in
the germline cells of later stages.
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BicD and the Dynein complex are required for meiosis and Polo localization
As Polo interacts with BicD and localizes to the oocyte, we tested whether
Polo localization is dependent on BicD and we compared this localization to
meiosis progression. First, germline clones of an amorph BicD allele
(BicDr5) in flies expressing GFP-Polo showed a staining
for the SC component C(3)G in all cells of a cyst in region 2a
(Fig. 3A). However, this
staining was weaker than that observed in pro-oocytes of wild-type cysts and
did not have the typical morphology of wild-type SCs even if thread-like
structures were observed. C(3)G was no longer detectable in regions 2b and 3.
This reveals that, in the complete absence of BicD, all cystocysts enter
meiosis but do not progress to the full pachytene and instead revert back to
an endoreplicative nurse cell fate. A similar phenotype was observed in the
absence of the GFP-Polo transgene, indicating that entry into meiosis in the
absence of BicD was not due to overexpression of Polo (data not shown). This
result differs from a previous report in which the absence of another SC
epitope in BicDr5 clones led the authors to conclude that
BicD was required to initiate SC formation
(Huynh and St Johnston, 2000).
This suggests that during meiosis this unknown protein is recruited later to
the SC than C(3)G. In BicDr5 clones, Polo has a normal
spotted distribution in region 1 of the germarium
(Fig. 3A), indicating that this
peculiar subcellular localization of Polo is independent of BicD. However,
GFP-Polo dots were found in all the cells of the cysts in region 2a
(Fig. 3A), and they became
undetectable in regions 2b and 3 instead of accumulating in one cell of the
cyst.
We also analyzed GFP-Polo localization and meiotic progression in the
hypomorphic mutant BicDPA66. The resulting mutant BicD
protein retains some function but fails to localize and accumulate in the
presumptive oocyte, leading to the formation of cysts containing 16 nurse
cells (Suter and Steward,
1991) (Fig. 6A). In
region 2a of BicDPA66 germaria, meiosis initiated properly
in two to four cells per cyst indicating that a detectable active transport of
BicD protein is not required for this process
(Fig. 3B). Then, in region 2b,
the number of SC-positive cells varied from 0 to 2 depending on the cyst, but
we rarely observed cysts with only one meiotic cell (2/31). Cysts positive for
SCs in region 3 were an exception (see below). This strongly suggests that
BicD is required in the presumptive oocyte for the normal restriction of
meiosis to this cell. In the BicDPA66 mutant, GFP-Polo
dots failed to properly accumulate in the central part of the cysts in regions
2a and 2b, and were not found in the presumptive pro-oocyte. Polo was not
detected in region 3 of BicDPA66 germaria. Among 186
analyzed BicDPA66, only one contained GFP-Polo dots in a
cyst of region 3 and it was also the only one that had SC-positive cells (data
not shown).
BicD function during oogenesis is also dependent on Egl and the Dynein
complex (Mach and Lehmann,
1997; Bolivar et al.,
2001). To confirm that Polo localization depends on BicD function
in polarized transport, we also investigated Polo localization in an
egl null background. Polo localization and meiotic progression always
showed the same defects as in BicD null mutants
(Fig. 3C). We also generated
germline clones for a null mutant of the Dynein complex component
dynamitin (dmn)
(Januschke et al., 2002). In a
similar way to BicD and egl loss-of-function mutants, the
absence of Dmn resulted in Polo failing to accumulate in one cell of the cyst
(Fig. 3D). In most of the
dmn clones, meiosis started normally in four cells and was then
restricted to the two pro-oocytes in region 2a. However, dmn cysts in
region 2b contained 0, 1 or 2 C(3)G-positive cells, similar to
BicDPA66 ovaries (Fig.
3D). Meiosis was never observed in region 3 and cysts
systematically failed to form an oocyte. Together, these results show that
Polo localization and the restriction of meiosis to the oocyte are progressive
processes throughout region 2a, and that both are dependent on a polarized
transport to the oocyte.
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). This allele was associated in trans with a chromosomal
deficiency covering the polo locus (Df(3L)rdgc-cos2) or the
strong alleles polo9 or polo16-1
(Donaldson et al., 2001). These
three different genotypes gave identical phenotypes that were completely
rescued by one GFP-Polo transgene. In polo cysts, C(3)G staining was
usually found in only a few spots in each cyst in region 2a
(Fig. 4B,B'). By
contrast, cysts in region 2b contained two to four cells with normal SCs
reaching the pachytene, which in wild type is typical for region 2a,
indicating a significant delay in meiosis entry and in the restriction to one
cell. C(3)G was still present in at least two cells in region 3, thus
confirming the delay of meiosis restriction to one cell
(Fig. 4B,B'). However,
the staining intensity for C(3)G is reduced in region 3 compared with wild
type, and the protein was only found in a few small dots per nucleus.
Surprisingly, in later stages, meiosis was restricted to the oocyte and the
SCs appeared normal. In conclusion, partial loss of Polo function led to two
distinct phenotypes during the first steps of meiosis. On the one hand, it is
involved in the initiation of SC formation and in their maintenance in the
oocyte. On the other hand, it is also involved in the restriction of meiosis
to the oocyte. We also investigated oocyte differentiation in polo mutants using the BicD protein itself as a reporter. In wild-type conditions, BicD started to accumulate in the pro-oocytes as early as region 2a, and was globally restricted to the future oocyte when the cysts entered region 2b (Fig. 4A,A''). When the cysts progressed into region 3, BicD migrated from the anterior to the posterior margin of the oocyte, indicating its antero-posterior polarization. In cysts with a partial loss of Polo function, BicD failed to accumulate in pro-oocytes of region 2a (Fig. 4B,B''). However, the accumulation of BicD was only delayed, as it started properly in region 2b. In region 3, BicD remained at the anterior of the oocyte, but this polarization defect was corrected in later stages indicating that it corresponds to a delay in oocyte differentiation. We did not observe important changes in microtubule organization in polo mutant cysts suggesting that Polo does not act through a direct effect on the microtubule network. However, DNA and BicD staining revealed that, in less than 1% of cases, hypomorphic polo mutations led to cysts without an oocyte and with 16 endoreplicative nurse cells, confirming that polo is involved in meiosis and oocyte differentiation (data not shown).
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Polo overexpression affects meiosis progression and oocyte differentiation
As loss of Polo function seems to indicate that Polo is required for
meiosis, we wondered whether Polo might act as a trigger for meiosis when
overexpressed. Polo overexpression in the germline was obtained in two ways.
On the one hand, we used flies that were homozygous for the GFP-Polo transgene
in a wild-type context for endogenous polo. On the other hand, we
produced flies in which a UAS-polo construct was specifically expressed in the
germline. Similar phenotypes were observed in both lines. First, in region 2a
we observed that approximately half of the cysts had more than four cells
containing SC with, generally, six to eight cells in meiosis
(Fig. 5A). Thus, Polo
overexpression can induce more cells of a cyst to enter meiosis than is seen
in the wild type. Furthermore, in regions 2b and 3, cysts always contained at
least two cells with SC. In some cases, cysts in region 3 still contained four
C(3)G-positive cells (Fig. 5).
The restriction of meiosis to one cell eventually occurs during stages 3-5.
Observation of Polo distribution itself gave further insight into this
phenotype. Intense spots of GFP-Polo were observed in more than one cell per
cyst, even in regions 2b and 3 (Fig.
5B,B'''). Moreover, the presence of intense GFP-Polo spots
correlated with the presence of C(3)G-positive cells. Finally, Polo became
restricted to the oocyte at the same time as meiosis during vitellogenic
stages. Surprisingly, increased Polo function led to defects in oocyte
differentiation similar to those caused by partial loss of function: delay in
the accumulation of BicD in the oocyte and early polarization of the oocyte
(Fig. 5B''). These results
show that Polo overexpression leads to a delay in its own localization to the
oocyte, probably because its overabundance exhausts the process leading to its
asymmetric distribution. Polo overexpression also induced defects in the
initiation and restriction of meiosis, and these defects correlated with Polo
localization. As in the case of partial loss of polo function, these
data strongly suggest that Polo is involved in the initiation, maintenance and
restriction to one cell of meiosis. Our data suggest that meiosis is
controlled by the level of the Polo protein in each cell of the cyst, and that
the specific localization of Polo to the oocyte is required for meiosis
restriction.
|
).
BicDPA66 shows a decreased level of phosphorylation, which is
probably responsible for its reduced functional activity. When we
overexpressed a UAS-Polo in a BicDPA66 mutant germline, we
observed that the follicles contained an oocyte at their posterior
(Fig. 6B,C), and that SCs were
present in every cyst from region 2 of the germarium until stage 6 follicles
(Fig. 6B). All of the control
BicDPA66 follicles examined (n=224) had 16 nurse
cells, whereas 98% of BicDPA66 follicles overexpressing
Polo (n=238) had an identifiable oocyte. BicD localized
preferentially to the posterior of the oocyte in these follicles
(Fig. 6B), although not as well
as in wild-type follicles. Eventually, this posterior localization of BicD was
not maintained beyond stages 3 or 4 of oogenesis, and germ cells of these
cysts degenerated at about stage 8 of oogenesis
(Fig. 6C). As BicD localization
is dependent on its own function in polarized transport, these data indicate
that overexpression of Polo in the germ cells is able to suppress the early
phenotypes of BicDPA66 and to restore its ability to
mediate polarized transport to the oocyte. This confirms that Polo has a
direct role in regulating the polarized transport in germline cells and
suggests that this function is mediated by BicD. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Polo localization to the oocyte requires BicD-dependent polarized transport
This paper describes the localization of the Polo protein and its genetic
control in the Drosophila germline during early oogenesis. Polo has a
peculiar subcellular localization in cytoplasmic dots that do not correspond
to any well-known structures of germline cysts or to microtubule minus-ends
where BicD accumulates. Polo has previously been described as colocalizing
with several subcellular structures depending on cell cycle phase
(Barr et al., 2004), but none
of these corresponds to the localization observed in the present study.
Similar cytoplasmic dots were observed in the primordial germline cells of the
Drosophila embryo as soon as they were formed, suggesting that this
unusual localization could be a specific feature of the germline
(Moutinho-Santos et al.,
1999).
From region 2a onward, Polo dots are present mostly in the cells containing SCs. This is the first report of a cell-cycle regulator whose localization is spatially and temporally correlated with meiotic progression during early oogenesis. Moreover this correlation is still conserved in mutants that affect polarized transport and the restriction and maintenance of meiosis. This indicates that Polo localization is dependent on polarized transport. One possibility is that Polo itself is directly transported to the oocyte. This hypothesis is reinforced by the physical interaction between BicD and Polo proteins, according to the proposed function of BicD as adapter for Dynein cargos. However, the BicD-dependent localization of Polo is not sufficient to explain its expression profile. Polo is strongly expressed in region 1 of the germarium, and the overall amount of the protein in the cyst progressively decreases, becoming undetectable after stage 2. This degradation seems to be compensated in meiotic cells and then in the oocyte by the polarized transport. The progressive degradation of Polo is also observed in egl and BicD null mutants. Degradation in association with a complete absence of Polo transport may explain why all the cells of a cyst enter into meiosis in these mutants (all the cells contain the same amount of Polo), and then exit meiosis simultaneously (none of the cells preferentially accumulates enough Polo). Alternatively, rather than by direct transport of Polo to the oocyte, its asymmetric distribution in the cyst could be due to a differential control of its stability between nurse cells and oocyte under the control of the BicD-dependent polarized transport.
|
). In contrast to BicD, Dynamitin is not involved in the
initial restriction of meiosis, showing that the interaction of BicD with
Dynamitin, and thus probably Dynein, is not required for the initial
restriction of meiosis. In a similar way, LC8 null mutants or
egl mutants that specifically block the interaction between Egl and
LC8 do not interfere with the initiation of meiosis in only four cells
(Navarro et al., 2004). We
found that transport of the BicD protein between the cyst cells is apparently
not required for this first step, as the BicDPA66 allele
or drug-induced microtubule depolymerization does not affect this initial
restriction, although BicD is diffuse throughout the entire cyst (see Results)
(see also Huynh and St Johnston,
2000). Finally, a null mutant for the plakin shot, which
has been proposed to be an essential upstream component of the Dynein function
in centrosome migration, exhibits variable meiotic phenotypes but allows a
normal initial restriction of meiosis to four cells
(Roper and Brown, 2004). These
data are consistent with a function of BicD and Egl independent of Dynein in
the initial restriction of meiosis.
Polo is involved in the control of meiosis
Polo is involved in many crucial steps of the cell cycle, including the
G2/M transition of mitosis and meiosis processes (reviewed by
Barr et al., 2004). Here, we
show that hypomorphic polo alleles lead to a delay in meiotic entry
and that Polo overexpression can trigger meiosis in more than four cells per
cyst in region 2a. These phenotypes could be related to the function of Polo
in the G2/M transition. In vertebrates, Polo is an activator of the
String/CDC25 phosphatase, and it has also been proposed that Polo can repress
the kinases Myt1 and Wee1. String is the main activator of the cyclinB/CDC2
complex, the activity of which triggers the G2/M transition, whereas Myt1 and
Wee are repressors of this complex. However, the role of the cyclin B and
CDC25 in meiosis in Drosophila oogenesis is not yet well understood
because, for example, CDC25 seems to act as a negative regulator of meiotic
oocyte cell fate (Mata et al.,
2000). Further investigations will be needed to determine how Polo
triggers meiotic entry during early oogenesis.
We have shown that in mutants with partial loss of polo function,
SCs start to disassemble in region 3 but are well formed again in stage 2/3
before disappearing in the following stages. One possible hypothesis to
explain how meiosis is finally properly maintained in polo
hypomorphic mutants is that the repression of cyclin E by Dacapo during stage
2/3 represses endoreplication, and thus allows meiotic progression
(Hong et al., 2003). This is
consistent with the finding that the specific localization of Dacapo to the
oocyte and its requirement for meiosis maintenance begins only in region 3.
Moreover, null mutations of dacapo do not lead to a fully penetrant
16-nurse-cell phenotype, confirming the existence of a partially redundant
control. Therefore, we propose that the balance in favor of meiosis is
initially due to the localized activation of meiosis by Polo, and later to the
localized inhibition of endoreplication by Dacapo, and that both mechanisms
partially overlap.
|
).
However, at least two results argue for a direct role of Polo in polarized
transport, independently of its meiotic function. First, in mosaic germline
cysts, nonmeiotic cells mutant for polo retain BicD protein. Thus,
this phenotype cannot be due to the activation of the meiotic checkpoint. This
strongly suggests that Polo is required in each cell of the cyst to initiate
BicD-dependent transport to the presumptive oocyte. Second, the overexpression
of Polo is able to restore the localization and therefore the function of
BicDPA66 protein. Interestingly, this mutant allele is due to a
single amino acid substitution (A40V) that leads to a hypophosphorylation of
BicD, and genetic evidence indicates that this phosphorylation is crucial for
BicD function (Suter and Steward,
1991). Polo overexpression might restore a functional level of
BicDPA66 phosphorylation. Therefore, even if we failed to observe
significant change in the gel mobility of BicD in polo hypomorph
mutants, it is tempting to propose that the function of Polo in the polarized
transport could be to activate, directly or indirectly, BicD by
phosphorylation.
A model for oocyte determination and meiosis control
Taken together, our results lead us to propose a model that can explain a
reciprocal requirement between the control of meiosis and oocyte specification
(Fig. 6C). This model is based
on four major points. First, BicD is required for the Dynein-dependent
polarized transport of oocyte determinants. Second, BicD is also required for
the progressive localization of Polo to the oocyte. Third, Polo appears to
trigger meiosis in the germarium. Fourth, Polo is required to activate the
BicD and Dynein-dependent polarized transport. These findings suggest the
existence of a positive feedback loop between Polo and BicD proteins, and
therefore between oocyte specification and meiosis.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Barr, F. A., Sillje, H. H. and Nigg, E. A.
(2004). Polo-like kinases and the orchestration of cell division.
Nat. Rev. Mol. Cell Biol.
5, 429-440.[CrossRef][Medline]
Bolivar, J., Huynh, J. R., Lopez-Schier, H., Gonzalez, C., St
Johnston, D. and Gonzalez-Reyes, A. (2001). Centrosome
migration into the Drosophila oocyte is independent of BicD and egl, and of
the organisation of the microtubule cytoskeleton.
Development 128,1889
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