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First published online 19 December 2007
doi: 10.1242/dev.016253
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Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA.
* Author for correspondence (e-mail: wumin{at}bio.fsu.edu)
Accepted 6 November 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Lethal (2) giant larvae (Lgl), Atypical protein kinase C (aPKC), Oocyte polarity, Par-1, Microtubule, Oogenesis, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Drosophila oocyte exhibits an anteroposterior (AP) asymmetry
with respect to its cytoskeletal organization and the distribution of some
mRNAs and proteins (Riechmann and
Ephrussi, 2001
; van Eeden and
St Johnston, 1999). This asymmetry provides the basis for the
formation of the major body axes in the subsequent embryo. A key step for
oocyte polarity formation is the reorganization of the microtubules in the
germline during mid-oogenesis. Previous studies show that the microtubule (MT)
nucleating activity is associated with the centrosome-nucleus complex
(Januschke et al., 2006).
After stage 6, MT reorganization in the oocyte occurs at the onset of anterior
migration of the nucleus (Januschke et
al., 2006), presumably as a consequence of the polarizing signal
from the follicle cells (Gonzalez-Reyes
and St Johnston, 1998; Roth et
al., 1995; Ruohola et al.,
1991). Reorganization of the posterior microtubule organizing
center is believed to allow formation of a gradient of MT from high density at
the anterior to low density at the posterior
(Cha et al., 2001;
Januschke et al., 2006;
Theurkauf et al., 1992). When
the reorganization of MT within the oocyte takes place at this stage, the MT
plus ends, as visualized by the localization of a protein fusion of the
plus-end MT motor kinesin (Kin:β-Gal)
(Clark et al., 1994),
accumulate in the posterior. This new microtubule orientation is necessary for
localization of maternal determinants such as oskar (osk),
bicoid (bcd), and gurken (grk) mRNAs,
which are the foundation for specification of the AP and dorsoventral axes
(Neuman-Silberberg and Schupbach,
1993; Nilson and Schupbach,
1999; van Eeden and St
Johnston, 1999).
Lethal (2) giant larvae (Lgl), a WD40 domain-containing protein, is
implicated in cellular asymmetry formation in a number of cell types
(Vasioukhin, 2006;
Wirtz-Peitz and Knoblich,
2006), but its role in the oocyte is unclear. Lgl is
evolutionarily conserved across the eukaryotes; orthologs range from budding
yeast (Sro7/Sro77) to human (Lgl1 and Lgl2)
(Vasioukhin, 2006). In
Drosophila, lgl mutations result in loss of apicobasal polarity in
epithelial cells and produce tumors of the brain, the imaginal disc and the
follicular epithelium (Bilder et al.,
2000; Ohshiro et al.,
2000; Peng et al.,
2000). In neuroblasts, Lgl is phosphorylated by aPKC in the apical
region to direct localization of basal components involved in asymmetric cell
divisions (Betschinger et al.,
2003). The precise role of Lgl in cell polarity formation in
Drosophila and other multicellular organisms remains largely unknown.
Here, we report that Lgl and its phosphorylation by aPKC are required for
oocyte polarity formation. aPKC phosphorylation of Lgl restricts Lgl activity
to the oocyte posterior and regulates posterior enrichment of Par-1 and
organization of microtubule polarity that is required for morphogen
localization and axis specification.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Manfruelli et al.,
1996; Ohshiro et al.,
2000). lgl and apkc germline clones were
generated by FLP-FRT-induced mitotic recombination
(Xu and Rubin, 1993), and
lgl germline clones were also generated by the DFS
FRT/FLP-ovoD1 system (Chou and
Perrimon, 1996) from the following strains:
lgl4P[neoFRT]40A/Cyo (strong LOF allele),
lgl4w3P[neoFRT]40A/Cyo (strong LOF allele),
P[ry+, FRT]42D
apkcK06403/Cyo (strong LOF allele), yw
P[mini-w+, hsFLP]1; P[Ubi-GFP]33
P[Ubi-GFP]38 P[neoFRT]40A/CyO, yw
P[mini-w+, hsFLP]1; P[ovoD1-18]2La
P[ovoD1-18]2Lb P[neoFRT]40A/CyO, yw
P[mini-w+, hsFLP]1; P{arm-lacZ.V}36BC
P[neoFRT]40A/CyO, w[1118]; P[neoFRT]42D P{w[+mC]=Ubi-GFP(S65T)nls}2R/Cyo. We
induced germline clones by administering 2 hour 37°C heat shocks on two
consecutive days during the third instar. For aPKC and GFP-Par-1(N1S)
overexpression, we obtained UAS:aPKC and
UAS:aPKC
N transgenic fly stocks
from C. D. Doe (Lee et al.,
2006), transgenic fly stocks with pUASP: GFP-Par-1(N1S)
on the 2nd chromosome from D. St Johnston
(Huynh et al., 2001), and
transgenic fly stocks with pUASP:GFP-Par-1(N1S) K* and
pUASP:GFP-Par-1(N1S) on the 3rd chromosome from A. Ephrussi
(Vaccari et al., 2005).
Mat
4-tubulin-Gal4-VP16 (Mat-Gal4) was used
for the overexpression in the germline.
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) and
apkc from DGRC were amplified, sequenced and subcloned into the pUASP
(Rorth, 1998) or pUASP:GFP
vectors for Drosophila transgenes. Transgenic lines of (1)
pUASP:Lgl, (2) pUASp:Lgl-3A, pUASP:Lgl-GFP, (3)
pUASP-Lgl-3A-GFP, (4)
pUASP:aPKC-GFP and (5)
pUASP:aPKCN-GFP
constructs were generated by standard methods and overexpressed by means of
the germline Gal4 drivers (Mat-Gal4). One of each of these
transgenic lines were used for phenotypic and biochemical analyses.
For the co-immunoprecipitation (co-IP) analysis, ovarian extracts were
prepared from wild-type flies or transgenic flies expressing pUASP:GFP-Par-1
(N1S) in homogenization buffer [150 mM NaCl, 50 mM HEPES (pH 7.2), 0.5 mM DTT,
PMSF, Complete Protease Inhibitor cocktail (Roche)]. Lysate was incubated with
primary antibody (rabbit anti-Lgl antibody)
(Betschinger et al., 2003) or
mouse anti-GFP antibody (Santa Cruz Biotechnology) for 10 hours at 4°C.
Protein A/G plus-agarose (Santa Cruz) was then added, and the reaction was
allowed to proceed for 4 hours at 4°C. Analysis was conducted with
SDS-PAGE followed by western blots. In anti-Lgl immunoprecipitates, controls
were only beads (without added antibody) and peptide blocks; in anti-GFP
immunoprecipitates, controls were beads (without added antibody) and the
ovarian extract from wild-type flies. Anti-Cut antibody was used as a negative
control in the co-IP experiment, which did not detect the Cut band in the
western blot containing proteins pulled down by anti-Lgl antibody (data not
shown).
Antibody staining, imaging and analysis
Antibody staining was performed according to standard procedures
(Sun and Deng, 2005). Primary
antibodies were diluted as follows: rabbit anti-Lgl and anti-pLgl, 1:200
(Betschinger et al., 2003);
rabbit anti-aPKC, 1:1000 (Santa Cruz Biotechnology); rabbit anti-Stau, 1:5000
(St Johnston et al., 1991);
mouse anti-Gurken, 1:20 (Developmental Studies Hybridoma Bank (DSHB)); rabbit
anti-β-galactosidase, 1:5000 (Sigma); Alexa Fluor 546 Phalloidin, 1:50
(Invitrogen), mouse anti-Cut, 1:50 (DSHB); mouse anti-Hindsight (Hnt), 1:20
(DSHB); mouse anti-Eyes absent (Eya), 1:50 (DSHB). For microtubule staining,
samples were fixed for 10 minutes in 8% paraformaldehyde
(Doerflinger et al., 2006) and
stained with a FITC-conjugated monoclonal anti--tubulin antibody DM1A
(1:400; Sigma). Secondary antibodies conjugated to Alexa Fluor 546 goat
anti-mouse and goat anti-rabbit (Molecular Probes) were used at 1:400.
Fluorescently labeled samples were counterstained with DAPI for visualization
of DNA. In situ hybridizations were carried out with RNA probes labeled with
Digoxigenin-UTP (Roche). Immunohistochemical detection was performed with
alkaline phosphatase-conjugated anti-DIG (1:5000; Roche). Images were captured
with a Zeiss LSM 510 confocal microscope and assembled in Adobe Photoshop.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Stau, an
RNA-binding protein that colocalizes with osk mRNA to the oocyte
posterior (St Johnston et al.,
1991) (Fig. 1A,E),
showed abnormal localization in lgl4 germline clones at
stages 9/10 (58%, n=81; 17% mislocalized to the center (hereafter
called `complete mislocalization'), 31% had both ectopic and posterior
localization ('partial mislocalization') and 10% had diffuse mislocalization
('diffuse mislocalization'); Fig.
1B and data not shown). The TGF- homolog Grk, which
normally moves with the oocyte nucleus from the posterior to an anterior
corner to induce dorsoventral patterning
(Neuman-Silberberg and Schupbach,
1993) (Fig. 1C),
failed to reach its correct destination in some lgl4
germline clones (16%, n=144; Fig.
1D). In these cases, Grk and the oocyte nucleus were either
mislocalized to a lateral position in the oocyte
(Fig. 1D) or remained at the
posterior (data not shown). To confirm the involvement of Lgl in localizing
oocyte polarity markers, we generated germline clones of a different
lgl mutant allele, lgl4w3, and similar defects of
Stau and Grk localization were found (data not shown). When RNA in situ
hybridization was used to stain the lgl4 germline clones
generated by a dominant female sterile (DFS) technique, the posterior
determinant osk mRNA (Kim-Ha et
al., 1991) (Fig.
1E) was mislocalized in the oocyte at stages 9/10 (68%,
n=143; 13% complete, 38% partial and 17% diffuse;
Fig. 1F and data not shown).
bcd mRNA, which is normally localized to the extreme anterior cortex
of the oocyte in a disc-shaped pattern
(Driever and Nusslein-Volhard,
1988) (Fig. 1G),
accumulated as dots in the anterior of lgl germline clones (38%,
n=128; Fig. 1H), also
indicating a defect in localizing the anterior determinant.
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), which is
normally localized at the posterior end of the oocyte during stages 8-9
(Fig. 1I). In lgl
germline clones, Kin:β-gal was not concentrated at the posterior and
instead mislocalized as ectopic dots in the center or partially mislocalized
or diffused from the posterior of the oocyte (57%, n=97; 14%
complete, 36% partial and 7% diffuse; Fig.
1J and data not shown). In oocytes of lgl germline
mutants, the anterior-to-posterior gradient of microtubules at stages 7-8
(Theurkauf et al., 1992)
(Fig. 1K), as visualized by
direct staining with a FITC-conjugated antibody against -Tubulin, was
also disrupted. Instead of an AP gradient, a high density of microtubules was
detected throughout the oocyte of lgl germline clones
(Fig. 1L). These defects in
microtubule organization could contribute to the defects in Stau/osk
and Grk mislocalization.
Follicle-cell differentiation is normal in lgl germline clones
Because the reorientation of microtubule polarity in the oocyte depends on
a polarizing signal from the posterior follicle cells (PFC), we examined
whether these follicle cells were specified correctly in egg chambers bearing
lgl germline clones. First, we stained the lgl mosaics with
antibodies against Cut and Fasciclin 3 (Fas3), which are both downregulated by
Notch after stage 6 (Lopez-Schier and St
Johnston, 2001; Sun and Deng,
2005) (Fig.
2A,A' and data not shown). In the PFC covering the
lgl germline clones, we found no change in the expression of these
two immature follicle cell-fate markers
(Fig. 2B,B' and data not
shown). Hindsight (Hnt), a Notch target in follicle cells during mid-oogenesis
(Sun and Deng, 2007),
maintained its expression correctly in the PFC after stage 6
(Fig. 2C,C'), suggesting
Notch signaling is unaffected in lgl germline clones
(Fig. 2D,D'). We then
examined the expression of Eyes absent (Eya), which is normally expressed in
both immature follicle cells and the anterior follicle cells after stage 7
(Bai and Montell, 2002)
(Fig. 2E,E'), and found
no expression in the PFC covering the lgl germline clone
(Fig. 2F,F'), suggesting
that the PFC do not take either the immature or anterior follicle cell fate.
In addition, a PFC fate marker, Pointed-lacZ (Pnt-lacZ)
(Gonzalez-Reyes and St Johnston,
1998; Poulton and Deng,
2006), continued to be expressed in the PFC covering the
lgl germline clones (Fig.
2H,H'). The correct expression of the follicle cell-fate
markers in follicle cells over the lgl germline clone suggests that
the activation of the Notch and EGFR pathways is unaffected. Therefore, the
possibility that Lgl is involved in the germline signaling that induces
follicle-cell differentiation can be excluded. Together, these data indicate
that Lgl acts downstream of the polarizing signal from the PFC to specify the
oocyte AP axis.
Phosphorylation of Lgl by aPKC is critical for oocyte polarity
Lgl can be phosphorylated by aPKC at three conserved serine residues in the
central region of the protein, a process required for inactivation of Lgl
(Betschinger et al., 2005;
Betschinger et al., 2003).
Using the co-IP technique, we detected aPKC in the protein complex pulled down
by a polyclonal antibody against Lgl from the ovarian lysate
(Fig. 6E), suggesting that Lgl
and aPKC interact during oogenesis.
To determine whether the phosphorylation of Lgl by aPKC is required for
oocyte polarization, we generated UASP:Lgl and UASP:Lgl-3A (a mutated form of
Lgl in which all three aPKC phosphorylation sites were mutated to alanine)
(Betschinger et al., 2003)
transgenes, overexpressed them using a germline Gal4 driver
Mat4-Gal4-VP16 (Mat-Gal4), and examined
oocyte polarity markers in these egg chambers. To confirm the expression of
Lgl and Lgl-3A in the germline, we performed a western blot using the anti-Lgl
and anti-phosphorylated-Lgl (pLgl)
(Betschinger et al., 2003). We
found that levels of pLgl in ovaries with germline overexpression of the
wild-type Lgl transgene were more than twice as high as endogenous levels,
whereas only endogenous levels of pLgl and a similar level of Lgl were
detected in Lgl-3A-overexpressing egg chambers (see Fig. S1A,B in the
supplementary material). Therefore, levels of expression of Lgl in Lgl and
Lgl-3A overexpressing chambers were similar, as were levels of
phosphorylatable Lgl in wild-type and Lgl-3A overexpressing chambers. Stau and
osk mRNA were mislocalized in the majority of the
Lgl-3A-overexpressing egg chambers at stages 9/10 (Stau: 74%, n=149;
44% complete, 10% partial and 20% diffuse,
Fig. 3C,I; see Fig. S2C in the
supplementary material; and data not shown; osk: 80%, n=130;
46% complete, 15% partial and 19% diffuse;
Fig. 3F,I; and data not shown),
Grk mRNA in the oocyte nucleus was mislocalized in 8% (n=150) of
these egg chambers (Fig. 3L),
and bcd mRNA was mislocalized to the posterior in 5% (n=120)
of these egg chambers (see Fig. S3B in the supplementary material). In
addition, Kin:β-gal was mislocalized in the oocyte (77%, n=121;
54% complete, 13% partial and 10% diffuse;
Fig. 3O and data not shown),
and a high density of microtubules was detected around the cortex
(Fig. 3R) or in the central or
lateral part of the oocyte (data not shown), indicating severe defects in
microtubule organization in the oocyte. By contrast, although wild-type
Lgl-overexpression also caused some Stau or osk mislocalization, the
percentage was much lower (Stau: 31%, n=123; 10% complete, 17%
partial and 4% diffuse, Fig.
3B,H, and see Fig. S2B in the supplementary material; and data not
shown; osk: 32%, n=149; 9% complete, 20% partial and 3%
diffuse; Fig. 3E,H; and data
not shown), diffuse localization of bcd mRNA was detected at the
anterior (3%, n=125; data not shown), and no defect was apparent in
Grk localization (n=123; Fig.
3K). Microtubules appeared to be only mildly affected in these egg
chambers (Fig. 3Q), and
Kin:β-gal was modestly disrupted at the posterior (36%, n=117;
10% complete, 18% partial, and 8% diffuse;
Fig. 3N). Because the
differences between Lgl-3A and Lgl lie in whether they can be phosphorylated
by aPKC, the stronger oocyte polarity defects in Lgl-3A-overexpressing egg
chambers suggest that Lgl phosphorylation by aPKC is important for oocyte
polarization.
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). Because overexpression of Lgl-3A in the germarium using the
nanos-Gal4 drivers (Van
Doren et al., 1998; Wang and
Lin, 2004) did not cause any oocyte differentiation defect (data
not shown), other aPKC targets may be involved in the maintenance of the
oocyte fate during early oogenesis. Although 80% (n=101) of the
apkck06403 germline clones were arrested at stage 5 or 6,
we found that some germline clones developed until stages 9 and 10. In oocytes
of these clones, the posterior localization of Stau was disrupted (47%,
n=114; 35% complete, 7% partial and 5% diffuse;
Fig. 4B and data not shown),
suggesting a previously unidentified role for aPKC in polarizing the AP axis
of the oocyte during mid-oogenesis. In these apkc germline clones,
follicle-cell fate markers (Cut, Eya, and Hnt) showed normal expression
patterns (data not shown). These data suggest that aPKC and Lgl may function
in the same pathway to regulate the oocyte polarity. To determine whether apkc and lgl interact genetically in germline cells, we overexpressed Lgl in a heterozygous apkc mutant background. These egg chambers demonstrated a greater penetrance of oocyte polarity defects (Stau: 56%, n=128; 16% complete, 38% partial and 2% diffuse; Fig. 4C,D) than did those with Lgl overexpression alone, suggesting that Lgl and aPKC are functionally related in oocyte polarization. Moreover, co-overexpression of Lgl with aPKC alleviated the oocyte polarity defects (Stau: 10%, n=111; 1% complete, 8% partial and 1% diffuse; Fig. 4F,H) caused by Lgl overexpression alone (Fig. 4G and Fig. 3B), whereas overexpression of aPKC alone did not result in any obvious oocyte polarity defects (Stau: 96%, n=117; Fig. 4E). By contrast, co-overexpression of Lgl-3A with aPKC produced oocyte polarity defects similar to those of Lgl-3A overexpression alone (data not shown), suggesting that aPKC cannot modify the phenotypes caused by overexpression of the nonphosphorylatable form of Lgl. Together, these results strongly suggest that the phosphorylation of Lgl by aPKC plays a crucial role in regulating oocyte polarity formation.
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To determine the effect of phosphorylation of Lgl on its localization, we stained for Lgl protein in Lgl-3A-overexpressing egg chambers and found Lgl localization throughout the oocyte cortex (100%, n=153; Fig. 5E,F). This localization pattern was confirmed in another transgenic line with overexpression of GFP-tagged Lgl-3A (Lgl-3A-GFP) in the germline (Fig. 5G,H). The expansion of nonphosphorylatable and active Lgl along the oocyte cortex suggests that Lgl phosphorylation by aPKC plays an important role in restricting Lgl to the posterior of the oocyte.
To analyze further the relationship between Lgl phosphorylation and the
subcellular localization of Lgl in the oocyte, we monitored the distribution
of Lgl in egg chambers with co-overexpression of Lgl (or Lgl-GFP) and
aPKCN-GFP (or aPKCN) (a dominant active
form of aPKC) (Betschinger et al.,
2003). Most of these egg chambers failed to show Lgl association
with the oocyte cortex, and the rest showed only weak posterior or weak entire
cortex Lgl localization (no cortex localization: 59%; weak posterior: 23%;
weak entire cortex: 18%, n=87;
Fig. 5I,J and data not shown),
indicating Lgl is excluded from the cellular cortex by aPKC phosphorylation.
By contrast, the localization of Lgl-3A along the oocyte cortex was not
changed in egg chambers with co-expression of Lgl-3A and
aPKCN-GFP when compared with Lgl-3A overexpression alone
(see Fig. S4 in the supplementary material). These results suggest that
phosphorylation of Lgl by aPKC is crucial for Lgl localization in the
oocyte.
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;
Vaccari et al., 2005). Because
Lgl is also localized at the posterior cortex beginning at stage 7, we
performed co-IP experiments to determine whether Par-1 (N1S) and Lgl interact
biochemically in the germline cells. In the lysate from
GFP-Par-1(N1S)-overexpressing egg chambers
(Benton and St Johnston, 2003;
Huynh et al., 2001), Lgl and
GFP-Par-1 (N1S) immunoprecipitated together
(Fig. 6E), suggesting that Lgl
and Par-1 could form a complex in the germline cells during oogenesis.
To determine the relationship of Lgl and Par-1 localization in the oocyte,
we used both UASP:GFP-Par-1(N1S) and UASP:GFP-Par-1 (N1S) K*, a
kinase-dead form of the Par-1(N1S) isoform that has exactly the same
localization pattern as the normal Par-1 (N1S) protein
(Vaccari et al., 2005)
(Fig. 6B), to monitor any
change in Par-1 localization in lgl germline clones. We found that
both GFP tagged forms of Par-1 [(N1S) K* and (N1S)] were no longer
enriched at the posterior but became uniformly distributed in the cytoplasm
and cortical region of the oocyte (55%, n=31;
Fig. 6C,C' and data not
shown), suggesting that Lgl is required for Par-1 (N1S) enrichment at the
oocyte posterior. In egg chambers with Lgl-3A overexpression, active Lgl
expanded towards the lateral domains of the oocyte cortex. We asked whether
mislocalized active Lgl would disrupt Par-1 localization as well. In egg
chambers with co-expression of Lgl-3A and GFP-Par-1 (N1S) in the germline, we
found that GFP-Par-1 (N1S) expanded throughout the oocyte cortex, colocalizing
with Lgl-3A (Fig. 6D-D''),
indicating that active Lgl may play a role in directing Par-1 (N1S) to the
oocyte cortex.
Lgl regulates actin organization in the oocyte
Par-1 (N1S) localization depends on actin but not microtubule organization
in the oocyte (Doerflinger et al.,
2006), and GFP-Par-1 (N1S) showed tight association with the
cortical actin at the posterior cortex
(Fig. 7A). In egg chambers with
Lgl-GFP overexpression, Lgl-GFP also colocalized with the cortical actin
cytoskeleton (Fig. 7B).
Interestingly, under high magnification, the cortical actin in
Lgl-GFP-overexpressing egg chambers exhibited projections mainly at the oocyte
posterior (Fig. 7B). Careful
observation of wild-type oocytes also identified such actin-rich structures at
the posterior (Vanzo et al.,
2007) (84%, n=45; Fig.
7C), albeit weaker than those in the Lgl-overexpressing oocytes,
indicating that cortically localized Lgl may promote the formation of these
actin projections. To test this hypothesis, we monitored actin organization in
egg chambers with Lgl-3A overexpression or containing lgl4
germline clones. If Lgl function is involved in the formation of the actin
projections, the lateral oocyte cortex should have actin projections in egg
chambers with Lgl-3A overexpression, which have active Lgl localized along the
entire cortex. Indeed, in these egg chambers we observed long and thick actin
projections throughout the cortex of the oocyte (91%, n=55;
Fig. 7D). When treated with an
actin depolymerizing drug, latrunculin A, the actin projections were
dramatically compromised and mislocalized into the cytoplasm (data not shown).
By contrast, actin projections were undetectable in lgl4
germline clones (74%, n=35; Fig.
7E). These data suggest that active Lgl in the oocyte regulates
the organization of the cortical actin cytoskeleton.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Januschke et al., 2006;
Theurkauf et al., 1992). Our
findings show that genes such as aPKC and lgl that are
important in polarizing other cell types play important roles in regulating
the microtubule polarity in the oocyte. As in the neuroblast, aPKC regulates
Lgl function by affecting its localization along the cellular cortex
(Betschinger et al., 2003).
Lgl-3A, the nonphosphorylatable and active form of Lgl, was localized along
the oocyte cortex, whereas wild-type Lgl overexpression is mainly enriched at
the posterior. Lgl was less localized to the posterior cortex when it was
co-expressed with aPKCN. For establishment of the
posterior-to-anterior gradient of cortical Lgl, anterior aPKC activity is
probably stronger than at the posterior. Although preferential localization of
aPKC to the anterior cortex of the oocyte is plausible, similar to the apical
localization of aPKC in the cortex of the neuroblast or epithelial cells, we
did not observe such a strict anterior localization of aPKC in oocytes with
GFP-tagged aPKC overexpression or UAS-aPKC overexpression using anti-aPKC
antibody (data not shown). Although the transgene may not localize exactly the
same as the endogenous form, other mechanisms may also be involved in
restricting aPKC activity in the anterior so that Lgl is enriched at the
posterior oocyte cortex. For example, the mysterious signaling that is sent
from the posterior follicle cells might be involved in inactivating aPKC in
the posterior.
A previous report indicated that aPKC germline clones did not adversely
affect oocyte polarity in stage 9 egg chambers
(Doerflinger et al., 2006),
but in our experiments we did observe defects in oocyte polarity in some
apkc germline clones. Because no information was provided on the
allele used or the number of clones analyzed in the report
(Doerflinger et al., 2006), we
do not know why the polarity defect we observed was not detected in this
previous study. A possible reason is that not all apkc
(apkcK06403) germline clones that develop to stage 9/10
possess oocyte polarity defects, so a significant number of escapers have to
be analyzed before the phenotype can be observed.
The level or dose of Lgl and its phosphorylation by aPKC appear to be crucial for oocyte polarity formation. Both excessive and insufficient amounts of unphosphorylated Lgl cause oocyte polarity defects, probably explaining why overexpression of the wild-type Lgl also caused some defects in oocyte polarity, perhaps because of an upper limit on the amount of Lgl that can be phosphorylated by endogenous aPKC. When overexpression of Lgl is massive, only some of the overexpressed Lgl can be phosphorylated by aPKC, and the rest remains active and cortically localized. The alleviation of Lgl-overexpression-induced oocyte polarity defects by co-overexpression of Lgl and aPKC supports this hypothesis. A fine balance appears to be necessary between the amount of phosphorylated and unphosphorylated Lgl in the oocyte. Alteration of the levels of Lgl or aPKC can therefore cause varying severities of defects in oocyte polarity. Interestingly, lgl loss of function and Lgl-3A overexpression caused similar Stau/osk localization defects, which are probably results of disrupted actin and microtubule organization in these mutant backgrounds. The cytoskeletal defects could have been translated into defects in the Par-1 posterior enrichment that is crucial for Stau and osk localization.
Indeed, our data suggest that posterior enrichment of Par-1 is regulated by
Lgl. Par-1 has also been shown to be a phosphorylation target of aPKC in
HEK293 cells (Hurov et al.,
2004), and Par-1 localization could be regulated by aPKC
phosphorylation in the oocyte (Doerflinger
et al., 2006). aPKC therefore probably has dual function in
restricting Par-1 kinase activity to the oocyte posterior. First, it prevents
Par-1 from localizing to the anterior cortex through direct phosphorylation,
and second, it promotes Par-1 localization to the posterior by restricting
active Lgl to the posterior. To explain the function of Lgl in localizing
molecules in cell-polarity formation, two different, but not mutually
exclusive, models have been proposed
(Vasioukhin, 2006). The first
model is based on findings in yeast and vertebrate cells, demonstrating that
Lgl homologs regulate docking and fusion of post-Golgi vesicles with the
plasma membrane through the SNARE complex
(Lehman et al., 1999). In this
scenario, Lgl could be involved in the exocytosis pathway and membrane
trafficking. A sec5 mutant has been reported
(Murthy et al., 2005) to show
impaired membrane trafficking of Grk, but the cytoskeleton was correctly
oriented. These phenotypes differ from the defects we found in lgl
loss- and gain-of-function mutants. The potential involvement of Lgl in the
exocytosis pathway in the oocyte is therefore unlikely to be the sole
explanation of the serious polarity defects observed in
lgl4- and Lgl-3A-overexpressing egg chambers. The second
model is related to the role of Lgl in actomyosin cytoskeletal regulation,
which is based on genetic interactions between Lgl and actin-binding proteins
such as Myosin II [encoded by zipper (zip)] and Myosin VI
(Ohshiro et al., 2000;
Petritsch et al., 2003;
Strand et al., 1994). Our
finding that the cortical actin morphology was dramatically changed when the
active Lgl was overexpressed supports this model. In neuroblasts, Lgl can
promote the cortical localization of Miranda by restricting Myosin II
localization to the apical cortex. Germline clones of a zip
loss-of-function allele, however, caused arrest of oogenesis at an early
stage, before the reorganization of the microtubules takes place (data not
shown). By contrast, miranda loss-of-function germline clones showed
no defects in oocyte AP polarity (Irion et
al., 2006). A recent study demonstrated that actin projections at
the oocyte cortex require Osk (Vanzo et
al., 2007). Whether Lgl localization is normal in osk
mutants is unknown. Because osk is mislocalized in the Lgl-3A
overexpressing egg chambers, which still have long actin projections, the
impact of Osk in actin organization is probably indirect. Lgl and Osk may
affect some common targets that regulate cortical actin in the oocyte.
Our data indicate that genetic manipulations of either lgl or
apkc produced incomplete penetrance of oocyte polarity defects. In
fact, a number of previously reported mutations also had partial penetrance of
Stau mislocalization (14-3-3, 62%;
BAZS151A,S1085A misexpression, 49%; rab6,
64.47%), and the penetrance of mislocation of Grk/oocyte nucleus has not been
reported in these mutants (Benton and St
Johnston, 2003; Benton et al.,
2002; Coutelis and Ephrussi,
2007). Because many genes and pathways are involved in the
regulation of cytoskeletal organization in the oocyte, functional redundancy
and crosstalk between different pathways are probably unavoidable in this
complicated polarization process. Other genes may act in parallel with Lgl and
aPKC to regulate actin organization and Par-1 localization, which in turn
controls posterior localization of morphogens.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/3/463/DC1
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ACKNOWLEDGMENTS |
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