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First published online 30 November 2005
doi: 10.1242/dev.02179
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1 Institut Jacques Monod, Unité Mixte Recherche 7592, CNRS,
Université Paris 6 et Paris 7, 2 place Jussieu, 75251 Paris Cedex 05,
France.
2 Institut Curie, Section Recherche, Unité Mixte Recherche 144, CNRS,
75248 Paris, France.
Author for correspondence (e-mail:
guichet{at}ijm.jussieu.fr)
Accepted 25 October 2005
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin and Drosophila
pericentrin-like protein also strongly accumulate at the periphery of this
nucleus. MT polymerization after cold-induced disassembly in wild type and in
gurken mutants suggests that in the oocyte the centrosome-nucleus
complex is an active center of MT polymerization. We further report that the
MT network comprises two perpendicular MT subsets that undergo dynamic
rearrangements during oogenesis. This MT reorganization parallels the
successive steps in localization of gurken and oskar mRNAs.
We propose that in addition to a highly polarized microtubule scaffold
specified by the cortex oocyte, the repositioning of the nucleus and its
tightly associated centrosome could control MT reorganization and, hence,
oocyte polarization.
Key words: Microtubule organization, Nucleus, Centrosome, Polarity, Oocyte, Oogenesis, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The ovarian egg chamber consists of a
cluster of 16 interconnected germ cells, surrounded by a monolayer of
follicular epithelium. During egg chamber formation, one cell of the cluster
is singled out to become the oocyte, while its 15 sister cells develop into
nurse cells. This process of oocyte specification requires polar transport of
oocyte determinants mediated by minus-end directed MT motors (for a review,
see Palacios and St Johnston,
2001). MT nucleation is believed to be restricted to the posterior
of the oocyte from where MTs extend through the ring canals into the nurse
cells. The MT organization in the oocyte is unchanged until mid-oogenesis at
the onset of stage 7. Then, a signal emitted from the overlaying follicle
cells triggers the reorganization of the MT network and the migration of the
nucleus to the anterior. Subsequently, bicoid (bcd) mRNA is
localized to the anterior cortex of the oocyte. oskar (osk)
mRNA is transported to the posterior pole and gurken (grk)
mRNA is localized to an antero-dorsal cap near the oocyte nucleus (reviewed by
Riechmann and Ephrussi, 2001).
The localization of these axes determining transcripts occurs in several steps
and is dependent on MTs, cytoplasmic Dynein and Kinesin I. During the period
crucial for mRNA localization (stage 7-9), MTs are distributed in an
anteroposterior (AP) gradient, the highest concentration occurring in the
anterior part of the oocyte (Theurkauf et
al., 1993; Theurkauf et al.,
1992; Clark et al.,
1997; Micklem et al.,
1997). The orientation of MTs within this gradient was determined
using minus- and plus-end markers, i.e. Nod-ßgal and Kinesin-ßgal,
respectively (Clark et al.,
1994; Clark et al.,
1997). However, the use of these markers was not sufficient to
explain the successive MT-dependent localization steps of osk and
grk mRNA. Furthermore, the behavior of the microtubule organizing
center (MTOC) during oogenesis is complex, and the localization and nature of
the MTOC during this period are unknown. Shortly after oocyte specification,
nurse cell centrosomes migrate toward the oocyte, where they eventually
accumulate at the posterior of the nucleus
(Bolivar et al., 2001;
Mahowald and Strassheim,
1970). Centrioles reportedly disappear, as they could not be
detected after stage 4 (Mahowald and
Strassheim, 1970), whereas the PCM is still associated with the
oocyte nucleus, where -tubulin, Centrosomin and Pericentrin/AKAP450 can
be easily detected (Martinez-Campos et
al., 2004; Tavosanis and
Gonzalez, 2003). From stage 1 to stage 6, MTs are nucleated from a
posterior MTOC and at the end of stage 9, PCM components belonging to the
-TuRC complex have been found at the anterior and lateral cortices
(Cha et al., 2002;
Schnorrer et al., 2002).
However, during stages 7 to 8, the nature of the MTOC remains elusive. In the present work we have studied the nucleation and the organization of the MT network in the oocyte in order to better understand mRNA transport. We show that the association of a centriole-containing centrosome with the nucleus surrounded by PCM material constitutes an MTOC. Using a new fixation method that allows detection of essential components of the MT network and MTOC, we found that the oocyte contains two different MT populations. The two MT populations evolve differentially during oogenesis, which could constitute a scaffold for differential mRNA transport.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). MT extremities were detected using either
Kin-LacZ or Nod-LacZ (Clark et al.,
1997). Germ line clones for Khc were generated using
Khc7.288 FRT G13
(Januschke et al., 2002).
tubGAl4 is described in Januschke et al.
(Januschke et al., 2002). MTs
in living egg chambers were analyzed using polyubiquitin-GFPS65C-alphaTub84B
(Rebollo et al., 2004) and
Tau-GFP (Micklem et al.,
1997). The last 199 amino acids of the C-terminus of CG6735
(Kawaguchi and Zheng, 2004)
were amplified by PCR and cloned into the pUASpmGFP6 vector.
MT detection
MT detection was adapted from a method developed to preserve the
cytoarchitecture in cells in which antibody penetration is reduced
(Pizon et al., 2002). Ovaries
were incubated in BRB80 buffer (80 mmol/l PIPES pH 6.8, 1 mmol/l
MgCl2, 1 mmol/l EGTA), containing 1% Triton X100 (BRB-80-T) for 1
hour at 25°C without agitation. Then ovaries were fixed in MeOH at
–20°C for 15 minutes and rehydrated for 15 hours at 4°C in PBS
0.1% Tween, then blocked for 1 hour in PBS 0.1% Tween containing 2% (w/v)
bovine serum albumin (BSA) before incubation with primary antibody
overnight.
Immunohistochemistry and in-situ hybridization
Rabbit Anti-Khc AKIN02-A (Cytoskeleton, Denver, USA) 1:250, monoclonal
anti-
-tubulin (DM1A) FITC Conjugate (SIGMA) 1:250, rat Anti-tyrosinated
Tubulin YL 1/2 1:200 (Kilmartin et al.,
1982), mouse anti-polyglutamylated MTs on centrosomes, ID5
(Beisson et al., 2001). Rabbit
anti--tubulin: R46 [Tub37C
(Raynaud-Messina et al.,
2001)], 1:500 dilution, Rb1011 [Tub37C
(Tavosanis and Gonzalez,
2003)] 1:5, R77 [Tub23C
(Debec et al., 1995)] 1:500.
Mouse anti-ß-galactosidase (Promega), 1:200. Rabbit anti-PKCz C20 (Santa
Cruz Biotechnology, Inc.), 1:1,000. Anti-Osk and anti-Grk as described in
(Januschke et al., 2002).
PicoGreen (Molecular Probes) 3 µl/1 ml PBS 0.1% Tween for 45 minutes
following RNAse A treatment 0.4 µg/ml for 1 hour before staining. In-situ
hybridization was performed using grk- and osk-specific
probes according to Wilkie and Davis
(Wilkie and Davis, 2001).
Images were taken on a LEICA SP2 AOBS microscope (40x 1.25 NA oil),
except for Fig. 2C,D,F,
Fig. 3C,E,
Fig. 4F-H and
Fig. 5E-H, which were taken on
a Perkin Elmer Ultra View confocal scanner. Deconvolution was carried out
using the HUYGENS 2.6 software and the QMLE algorithm with seven iterations
and 3D reconstruction using the AMIRA 2.2 software. Living oocytes were
dissected in BRB buffer or M3 insect medium (Sigma). Electron microscopy
protocol is available upon request.
MT disassembly and regrowth
Complete depolymerization: ovaries were incubated for 30 minutes in
BRB-80-T at 25°C then placed on ice for 30 minutes and fixed. Complete
regrowth: ovaries were incubated for 30 minutes in BRB-80-T on ice, then
incubated at 25°C for 30 minutes and subsequently fixed. Partial regrowth:
ovaries were incubated in BRB-80-T at 25°C for 27, 25, 20 or 15 minutes,
placed on ice for 30 minutes and then transferred to 25°C for 3, 5, 10 or
15 minutes, respectively, then fixed and stained for MTs. Colchicine
treatment: flies were fed for 15 hours with colchicine (65 µg/ml) and
analyzed for MTs.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin has been shown to closely associate with the nucleus at the
posterior of the oocyte. In addition, electron microscopy studies have
demonstrated the presence of centrioles close to the oocyte nucleus up to
stage 4 (Mahowald and Strassheim,
1970). Thus, until stage 6, the centrosome associates with the
nucleus at the posterior of the oocyte. In Drosophila females,
meiosis takes place in the absence of centrosomes
(Mahowald and Strassheim,
1970). It has therefore been speculated that, at stage 6,
centrosome organization changes, involving the disappearance of centrioles and
the generation of MTs from a diffuse organizing center. To better understand
this process, we re-investigated the distribution of -tubulin in the
oocyte. Before repolarization of the MT cytoskeleton, we found that
Tub23C and Tub37C (Sampaio
et al., 2001; Sunkel et al.,
1995) localize in a layer around the nucleus, with an enrichment
at the posterior pole of the oocyte (Fig.
1A). This is in agreement with the location of the MTOC at this
stage. After repolarization of the MT cytoskeleton, both -tubulin
isoforms remain located in a perinuclear manner
(Fig. 1B,C). Interestingly,
Tub37C, but not Tub23C, labels a small body close to the oocyte
nucleus (Fig. 1, compare A,C).
In addition, Tub37C and Tub23C also exhibit differential
expression patterns in embryos: Tub37C is located with the centrosomes
of mitotic cells, whereas Tub23C is not
(Tavosanis and Gonzalez,
2003). Thus, -tubulin is distributed in close association
with the nucleus periphery and possibly on a centrosome-like structure.
Pericentrin/AKAP450 is another major component of the centrosome. Green
fluorescence protein (GFP) fusion of the C-terminal part of
Pericentrin/AKAP450 and its Drosophila homolog pericentrin-like
protein (D-PLP) have been shown to localize to the centrosomes respectively in
cultured human cells (Keryer et al.,
2003), Drosophila embryos and spermatocytes
(Martinez-Campos et al., 2004;
Rebollo et al., 2004). Using
the UAS/Gal4 system (Brand and Perrimon,
1993), we specifically expressed GFP-cter-D-PLP, in the germline
and detected a bright dot in the vicinity of the nucleus before and after
nuclear migration (Fig. 1D).
GFP-cter-D-PLP was also detected in all germline nuclei, as has been observed
previously (Martinez-Campos et al.,
2004). From stage 7 onward, the bright dot remained in the
immediate vicinity of the oocyte nucleus (<1 µm distance,
Table 1). Furthermore, both
GFP-cter-D-PLP and Tub37C co-localized to this discrete body,
indicating that this structure could correspond to a centrosome
(Fig. 1E-G). In G2 centriole,
tubulin is highly polyglutamylated
(Bobinnec et al., 1998). The
ID5 antibody labels basal bodies and centrioles in several species
(Beisson et al., 2001). Using
this antibody, we detected a dot close to the nucleus throughout oogenesis
that remained detectable up to stage 10A
(Fig. 1H). This suggests that
the dot represents a centriole-containing centrosome. Indeed, using electron
microscopy, (Fig. 1I,J), we
clearly detected two to possibly four centrioles closely associated with the
nucleus in stage 9 oocytes. This demonstrates the existence of centrioles
associated with the nucleus at least up to stage 9. We could not unambiguously
detect MT fibers emanating from those centrioles. We then analyzed the link
between centrosome and nucleus using colchicine. In flies fed with colchicine,
MTs in the germline were completely depolymerized, and the oocyte nucleus was
mispositioned (Fig. 1L). In the
oocyte, we observed that the nucleus and the centrosome were significantly
separated, the distance between them increasing during oocyte growth
(Fig. 1M,N and
Table 1). In a few cases, we
noticed that the nucleus could reach the anterior cortex without the
centrosome; however, we never observed a centrosome at the anterior without
the nucleus (Table 1). We
conclude that the close localization of the centriole-containing centrosome to
the nucleus depends on MTs.
|
;
Theurkauf et al., 1992).
In-between MT distribution has been described as an AP gradient
(Micklem et al., 1997).
However, high-resolution images of oocyte MTs are lacking. Therefore, we
modified for the Drosophila egg chamber a protocol frequently used to
increase the detection of the MT cytoskeleton in cell culture to characterize
MT organization in the oocyte during the crucial period in which bcd,
grk and osk mRNAs are localized. We detected MTs throughout
oogenesis using -Tubulin but also with a Kinesin heavy chain antibody
(-Khc), which revealed the MT array and its complexity in unprecedented
definition (Fig. 2A,B). We
noticed that the range of detected details was increased and more reproducible
with -Khc antibody. To control the specificity of Khc detection, we
generated germline and follicle cell mutant clones homozygous for
khc7.288. In such mutant cells, no Khc was detected
(Fig. 2B inset), indicating
that the detection is specific. Labeling with antibodies directed against
aromatic C-terminal amino acid residues (Tyr or Phe) of -tubulin
(Badin-Larcon et al.,
2004) and against Khc largely overlapped
(Fig. 2I,J). This confirmed
that the structures revealed by Khc were MTs. We also detected a Khc fraction
at the posterior of the oocyte (Fig.
2J), as it has been previously shown
(Brendza et al., 2002). That
Khc accumulates along MTs may be due to permeabilization before fixation,
which could cause rigor binding of Khc to MTs. Such case has been observed for
the motor XKCM1 without affecting MT organization itself
(Walczak et al., 1996). This
detection procedure may also permit the extraction of a soluble pool of Khc
and reveal the remaining fraction distributed along the MTs
(Hollenbeck, 1989). With our
detection procedure, Khc revealed by Kinesin-ßgal
(Clark et al., 1994) exhibited
a more restricted distribution compared with -Khc antibody
(Fig. 3H,I). This is probably
due to the substitution of the C-terminal part of Khc by the
ß-galactosidase in the reporter construct, impairing the recycling of the
chimeric Kinesin motor leading to its accumulation exclusively at the
posterior. We also show that with our detection method, the MT minus-end
marker, Nod-ßgal, was detected in the antero-dorsal corner above the
oocyte nucleus as well as in the opposite antero-ventral corner
(Fig. 3J,K), as it has been
reported before (Clark et al.,
1997; MacDougall et al.,
2003). Moreover, localized determinants such as Osk and Grk were
correctly positioned in the oocyte (Fig.
3L,M).
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).
Nevertheless, stretched follicle cells showed strikingly similar MT patterns
in living and fixed conditions as well
(Fig. 3B,C). Apicalbasal
polarity was not affected in follicle cells, as demonstrated by the correct
apical localization of atypical protein kinase C
(Fig. 3F,G). Importantly, the
MT distribution of living egg chambers expressing GFP--Tubulin at stage
7 (Fig. 2C) and stage 9
(Fig. 2F) was similar to the
one observed using anti--Tubulin and Khc antibodies
(Fig. 2D,E,G,H). Therefore it
seems that our fixation conditions preserve the wild-type MT organization and
that Khc can be suitable to label bulk MTs.
When fixed wild-type oocytes were analyzed by confocal microscopy, MT
organization in the oocyte appeared unchanged from stage 2 (data not shown) to
stage 6 (Fig. 4B, parts 1-5).
With stage 7 (
60%, n=11), MT organization was modified and two
MT subsets became apparent. This organization was more evident at stage 8
(69%, n=23). A first subset consisted of cortical MTs oriented
along the dorso-ventral (DV) axis parallel to the oocyte nurse cell border,
and juxtaposed to the lateral cortices, wrapping the oocyte from stage 7 to 9
(Fig. 4C-E, parts 1 and 5). At
least some MT bundles of this subset could be traced back to the oocyte
nucleus. The DV orientation of MT bundles, depicted as black fibers in the
schematic representations (Fig.
4C, part 6; Fig.
4D, part 6; Fig.
4E, part 6), was highly reproducible for all stages and persisted
throughout mid-oogenesis.
A second MT subset, depicted as red fibers in the schematic representations
(Fig. 4C, part 6;
Fig. 4D, part 6;
Fig. 4E, part 6), was present
in the center of the oocyte. Although there was some variability in the
patterns observed, we found that each developmental stage showed a
characteristic MT distribution. During stage 6 (82%, n=17), MTs
from this subset were cortical and extended from the nucleus at the posterior
to the anterior cortex (Fig.
4B, parts 2-4). At stage 7 (90%, n=11), compact
bundles of MTs formed a circle-like structure resembling a diaphragm. This
subset was formed by long MT bundles that extended (once or more) along the
entire cortex (Fig. 4C, parts
2-4). By stage 8 (65%, n=23), the oocyte had considerably grown
and individual MT bundles were therefore easier to track. MT bundles emanated
from the anterior and the nucleus to point toward the posterior
(Fig. 4D, part 2). MTs extended
again along the entire cortex, after which they turned to the central
cytoplasm (Fig. 4D, part 3).
This, in turn, generated free MT (plus) ends in the center of the oocyte
(Fig. 4D, part 4). By stage 9
(72%, n=18), the central MT network was clearly oriented along
the oocyte AP axis. One or two thick MT bundles extended from the anterior,
pointing toward the posterior pole. These bundles formed a structure
resembling a horseshoe, with its open side facing the posterior
(Fig. 4E, parts 2-4).
Importantly, both subsets could also be detected in living egg chambers, as
shown for the DV subset (Fig.
4F,H) and the AP subset (Fig.
4G). Thus, MTs show strong rearrangements throughout
mid-oogenesis, which results in two perpendicular MT arrays reflecting the two
axes of the oocyte.
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|
-Tubulin
distribution was not affected by cold-induced MT depolymerization
(Fig. 5C). When short periods
of regrowth were analyzed, MT nucleation appeared limited to the close
vicinity of the nucleus and was often asymmetric
(Fig. 5D,E), suggesting a
centrosome-associated nucleation activity. MT regrowth appeared to be
stepwise, as after 15 minutes only the DV cortical subset was established. MTs
clustered around the oocyte nucleus and aligned along the cortex in the DV
direction (Fig. 5F,G). The
cortical location of these fibers was clearly revealed by the presence of
Khc-positive dots at either the dorsal or the ventral side
(Fig. 5H). This indicates that
the DV MT subset is the first to regrow. We repeated the regrowth experiment
using colchicine. After the drug was washed out, MT repolymerization was
observed at the oocyte nucleus (data not shown). Taken together, these results
indicate that, at least with our detection method, the oocyte nucleus and its
immediate surroundings have the capacity to nucleate MTs.
The MT network is inverted in grk mutants
To test whether the centrosome-nucleus complex could direct the
repolarization of the MT network, we analyzed how MTs distribute in
grk mutant oocytes. In this mutant, the nucleus frequently remains at
the posterior of the oocyte due to a failure in the signaling cascade that
induces the repolarization of the cytoskeleton
(Gonzalez-Reyes et al., 1995;
Roth et al., 1995). In
grk mutant oocytes similar in size to wild-type stage 8, the MT
distribution was dramatically affected. Specifically, we found that MT
organization appeared completely reversed compared with wild type, in which
the nucleus is at the anterior and MT plus-ends are located toward the
posterior at stage 8 (Fig. 6A,B
versus Fig. 6E,F). In slightly
older oocytes, MTs remain stretched out along the cortex from the posterior
toward the anterior, where they fold back to the center of the oocyte
(Fig. 6B). MT ends in the
center are most probably plus-ends, as the pool of Khc (localized at the
posterior of wild-type oocytes, Fig.
3I) co-localizes with Kinesin-ßGal to the center of the
oocyte, between the flanking MT ends (Fig.
6D,G) (Clark et al.,
1994). Interestingly, MT distribution in grk oocytes was
strikingly similar to MTs of wild-type egg chambers before the migration of
the oocyte nucleus (Fig. 6E,F).
Likewise, the centrosome, as revealed by -tubulin, which is found at
the posterior of stage 6 wild-type oocytes, stays at the posterior in
grk mutants (Fig. 6H).
Thus, in grk mutants, distribution of MT and MTOC seemed similar to
their distribution in wild-type stage 6.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Bolivar et al.,
2001). Pericentriolar material closely associated with the oocyte
nucleus could be clearly detected until stage 6 with several centrosomal
markers, such as -tubulin, Centrosomin and D-Tacc
(Martinez-Campos et al., 2004;
Tavosanis and Gonzalez, 2003).
From stage 4 onward, the fate of the centriole cluster had been unknown
(Mahowald and Strassheim,
1970). In this study, we show that both Tub37C and
Tub23C are localized in a perinuclear manner throughout oogenesis.
Tub37C highlights a discrete body close to the nucleus. This body is
similarly detected by the centrosomal marker D-PLP and by a specific antibody
for polyglutamylated Tubulin, which detects centrioles. Consistent with this,
we have detected two to possibly four centrioles in the immediate vicinity of
the nucleus in stage 9 oocytes. This result demonstrates that at least until
stage 9, a centriole-containing centrosome is present in the oocyte.
Currently, we do not know whether they are still present at the onset of
meiosis I during stage 13, as it has previously been proposed that the meiotic
spindle is achieved without centrosomes
(Mahowald and Strassheim,
1970). During skeletal muscle morphogenesis, myotube centrosomes
dissociate from their nuclei, centrioles disappear and the centrosomal matrix
is redistributed to the nucleus periphery
(Tassin et al., 1985).
Similarly, during oogenesis, centrioles from nurse cell centrosomes may
disappear. However, their pericentriolar material may relocate to the oocyte
nucleus periphery. This would explain the specific enrichment of the oocyte
nucleus with perinuclear MTOC material. The only centrosome remaining
associated with a nucleus is that of the oocyte. Furthermore, the structure of
this centrosome remains intact. We conclude that the four centrioles found
close to the nucleus in stage 9 may correspond to the initial oocyte
centrosome in the duplication phase observed in G2.
|
;
Theurkauf et al., 1993;
Theurkauf et al., 1992) and
that MT minus-ends are associated with the cortex
(Cha et al., 2002), suggesting
that MTs could be nucleated there. With this work, we did not detect MT
nucleation at the lateral cortex, and MT disassembly and regrowth experiments
suggest that MT nucleating activity is associated with the centrosome-nucleus
complex. A lack of sensitivity in our MT detection method is unlikely to
explain the absence of nucleation from the oocyte cortex, as cortical MTs
could be detected in fixed egg chambers. Still, we cannot exclude that our
experimental procedures favor the regrowth from the oocyte nucleus. However,
only some MTs can be traced back to the nucleus surrounding (see Movie 1 in
the supplementary material), others only to the cortex. In addition, the MT
minus-end marker Nod-ßgal is detected at both corners of the anterior
cortex. Therefore we propose that MTs are nucleated at the centrosome-nucleus
complex, but only some of them are attached there; the remaining MTs might be
released. Such MTs would be subsequently translocated and captured at cortical
sites. MT translocation could involve actin filaments and motor proteins.
Interestingly, MT organization in the oocyte is affected when actin
organization is impaired (Manseau et al.,
1996). Such a mechanism has been observed in different cell types
(for a review, see Bornens,
2002). Hence, while MT nucleation is likely to be restricted to
the centrosome-nucleus complex, MT minus-ends are not. This would be in
accordance with the observation that injected bcd mRNA in the oocyte
accumulates at lateral cortices (Cha et
al., 2001).
We have observed that MT reorganization in the oocyte after stage 6 occurs
always at the onset of anterior migration of the nucleus, suggesting an
association between the nucleus and the MTOC. In agreement with this, a
mutation impairing the anchoring of the nucleus at the anterior cortex induced
a change in the MT network (Guichet et
al., 2001). Likewise, in grk mutant oocytes with the
nucleus at the posterior, the MT network seems organized as in wild-type stage
6 before MT reorganization. MT disassembly and regrowth suggest, at least with
our detection method, that a MT nucleating activity is associated with the
centrosome-nucleus complex, both in wild-type and in grk mutant
oocytes. Interestingly, laser ablation of the nucleus at the anterior of the
oocyte inhibited localization of determinants to the posterior, revealing a
failure of MT-dependent posterior transport in the absence of the nucleus
(Montell et al., 1991). These
results are in support of a role for the centrosome-nucleus complex in the
nucleation of the MT network necessary for correct polarized transport in the
oocyte. Interestingly, MT nucleation from the nuclear envelope, as well as
from centrosomes, has been described recently in Drosophila
spermatocytes (Rebollo et al.,
2004).
|
;
Schnorrer et al., 2002),
however, their MT nucleating activity has not been established. MT regrowth
experiments under our experiment condition, argue against such lateral MTOC
activity. MT nucleation activity of cortical MTOC components could be
repressed, for example by a Ran GTP gradient controlled by the nucleus. This
would ensure that centrosomal or non-centrosomal MTOC activity is restricted
to the vicinity of the nucleus. Such a mechanism has been reported for
Xenopus oocytes as well as for somatic cells
(Gruss et al., 2001;
Keryer et al., 2003).
A complex MT scaffold participates in axis specification
We are able to follow MT organization in the oocyte in high detail:
cortical MTs run in parallel to the DV axis throughout oogenesis, and a subset
of MTs oriented in the AP direction develops progressively in the center.
Using time-lapse images of GFP-Tubulin or Tau-GFP in live oocytes, we were
able to reproduce similar MT organization patterns to those observed with
fixed samples. The presence of two orthogonal subsets has been proposed
earlier (MacDougal et al.,
2003) and appears very probably adapted to specify different
compartments by facilitating differential mRNA transport
(Brendza et al., 2000;
Cha et al., 2002;
Duncan and Warrior, 2002;
Januschke et al., 2002;
MacDougall et al., 2003).
Indeed, there is a strong correlation between MT redistribution and mRNA
localization (Fig. 7).
grk and osk mRNAs show a dynamic and stage-dependent
localization pattern (Ephrussi et al.,
1991; Neuman-Silberberg and
Schuepbach, 1993). Both mRNAs are located at the posterior before
nuclear migration and move toward the anterior shortly after nuclear migration
and reorganization of the MT cytoskeleton. From stage 7 onward, grk
and osk mRNAs show striking differences in their localization (see
cartoon, Fig. 7). During stage
7, grk localizes along the anterior margin facing the nurse cell
border. During stage 8, grk is already restricted to the
antero-dorsal corner around the oocyte nucleus
(Fig. 7). This transport step
is dependent on MT motors (Duncan and
Warrior, 2002; Januschke et
al., 2002; MacDougall et al.,
2003). The DV subset, being fully established at stage 7, is a
candidate for such transport. This could further explain why grk mRNA
localization to the anterio-dorsal cap always precedes osk mRNA
localization to the posterior. Throughout the subsequent stages, as at least
some of the grk message arrives from the nurse cells, the DV MT
subset is likely to facilitate rapid transport toward the nucleus. Likewise,
osk mRNA localization to the posterior seems to be coupled to the
central MTs. osk localization occurs in a step-wise manner and is
only completed during stage 9. This correlates with the progressive
development of the center MT subset toward an AP orientation. We never
observed MT fibers touching the posterior cortex. Thus, osk mRNA
transport from the posterior-most ending of MTs obtained during stage 9 might
rely on other uncharacterized mechanisms.
During stages 7-9, bcd mRNA is transported to the oocyte, where it localizes as a ring in the anterior cortex. As it enters the oocyte, bcd mRNA encounters MTs necessary for its maintenance organized at the anterior cortex. In grk mutant oocytes, bcd localizes to the anterior as well as to the posterior pole. The anterior localization of bcd suggests the presence of MT minus-ends there. We did not detect MT nucleation activity at the anterior cortex in the cold-shock experiments. Therefore, posteriorly nucleated MTs might also in grk mutants be translocated to, and anchored at, the anterior cortex. However, we cannot exclude that we might have missed MT nucleation activity at the cortex due to the experimental setup.
Previous data suggest that MT distribution ranges from cortical enrichment
of MTs during stage 7 (Theurkauf et al.,
1992), via an intermediate step with MT plus-ends in the center
(Palacios and St Johnston,
2002), toward MT bundling, which promotes ooplasmic streaming late
in oogenesis (Theurkauf,
1994). The presence of perpendicular MT subsets has been proposed
(MacDougall et al., 2003).
Thus, MT organization changes dramatically during mid-oogenesis, but
stage-dependent configurations have not yet been established, and
high-resolution images of MTs were missing. Our results suggest different
stage-dependent MT configurations. This complex oocyte-wide network, in which
two MT subsets with a different spatial organization are apparent, could
provide a basis for MT motors to organize differential transport.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/1/129/DC1
* These authors contributed equally to this work 
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