1 Department of Molecular Biology and Genetics, Johns Hopkins University School
of Medicine, Baltimore, Maryland 21205, USA
2 Department of Molecular Biology and Genetics, Cornell University, Ithaca, New
York 14853, USA
* Author for correspondence (e-mail: gseydoux{at}jhmi.edu)
Accepted 6 December 2002
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SUMMARY |
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Key words: Polarity, Embryo, Par genes, C. elegans
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INTRODUCTION |
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In C. elegans, PAR-3, PAR-6 and PKC-3 are required to polarize the
newly fertilized egg along its anterior-posterior (AP) axis (for a review, see
Lyczak et al., 2002). This
axis is established shortly after fertilization and arises in response to a
cue associated with the sperm asters, which defines the posterior end of the
embryo. In response to this cue, the PAR-3/PAR-6/PKC-3 complex becomes
enriched on the cortex opposite the sperm asters (anterior side), while PAR-2
(a ring finger protein) and PAR-1 (a serine threonine kinase) become enriched
on the cortex nearest the sperm asters (posterior side). PAR-1 in turn is
required for asymmetric spindle positioning and for the asymmetric
localization of several developmental regulators
(Guo and Kemphues, 1995
). PAR-1
exerts its influence on developmental regulators through MEX-5 and MEX-6, two
redundant cytoplasmic CCCH finger proteins
(Schubert et al., 2000
).
The order in which these proteins function was determined by analyzing how
mutations in specific pathway components affect the localization of the other
components. For example, in par-2 mutants, PAR-3, PAR-6 and PKC-3
become delocalized and are found throughout the cortex
(Etemad-Moghadam et al., 1995;
Tabuse et al., 1998
;
Hung and Kemphues, 1999
);
similarly, in par-3, par-6 and pkc-3 mutants, PAR-2 is no
longer restricted to the posterior (Boyd et
al., 1996
). These effects have suggested that establishment of
anterior and posterior PAR domains depends on antagonistic interactions
between the PAR-3/PAR-6/PKC-3 complex in the anterior and PAR-2 in the
posterior. In contrast, mutations in par-1, mex-5 and mex-6
were reported not to affect PAR localization in the zygote
(Etemad-Moghadam et al., 1995
;
Boyd et al., 1996
;
Tabuse et al., 1998
;
Hung and Kemphues, 1999
;
Schubert et al., 2000
). These
mutations, however, have a dramatic effect on the distribution of several cell
fate regulators. For example, P granules and germline proteins, which in wild
type become restricted to the posterior end of the zygote, remain uniformly
distributed in par-1 mutants
(Kemphues et al., 1988
) and
mex-5;mex-6 double mutants
(Schubert et al., 2000
).
MEX-5, which in wild type localizes to the anterior, is mislocalized in
par-1 mutants, but PAR-1 localization is unaffected in
mex-5;mex-6 double mutants
(Schubert et al., 2000
). These
analyses have led to the `sequential repression model', whereby PAR-1
restricts MEX-5 to the anterior, and MEX-5 in turn restricts P granules and
germline proteins to the posterior
(Kemphues, 2000
). Whether
PAR-1 localizes MEX-5 by promoting its translocation to the anterior or by
negatively regulating its synthesis or stability in the posterior is not
known.
The actin cytoskeleton also plays a central role in the establishment of AP
polarity. The cortex of the zygote undergoes extensive contractions, which
drive internal cytoplasm towards, and superficial cytoplasm away from, the
sperm asters, creating a fountainhead effect
(Golden, 2000). Embryos treated
with cytochalasin D do not develop cortical contractions or cytoplasmic flow
and do not localize P granules or the germline protein PIE-1
(Hill and Strome, 1988
;
Hill and Strome, 1990
;
Reese et al., 2000
). Depletion
of components of the actomyosin network by RNAi (such as NMY-2, a non muscle
myosin II heavy chain, and MLC-4, a myosin light chain) also blocks flow and
delays P granule segregation (Guo and
Kemphues, 1996
; Shelton et
al., 1999
).
PAR proteins likely function intimately with the actin cytoskeleton to
polarize the zygote, but the details of this interaction remain poorly
understood. Mutations in par-2, par-3, par-5 and par-6 lead
to gene-specific defects in cortical contractions and cytoplasmic flow
(Kirby et al., 1990), and at
least one PAR protein (PAR-1) has been shown to interact directly with a
cytoskeletal component (NMY-2) (Guo and
Kemphues, 1996
). NMY-2 and MLC-4 depleted embryos, however, still
localize PAR-2 to a small region in the posterior cortex, raising the
possibility that the actin cytoskeleton is NOT required for the earliest steps
of polarization (Guo and Kemphues,
1996
; Shelton et al.,
1999
). The relationship between the sperm asters and the PARs is
also poorly understood. In particular it is not known (1) whether the polarity
cue associated with the sperm asters functions by recruiting PAR-2 or by
excluding the PAR-3/PAR-6/PKC-3 complex, and (2) how polarity is maintained
after pronuclear meeting when the sperm asters are no longer restricted to one
side of the embryo.
With only two exceptions to date (P granules and PIE-1)
(Hird et al., 1996;
Reese et al., 2000
), studies
describing asymmetric localization in the zygote have relied on
immunofluorescence experiments on fixed embryos. This method makes it
difficult to determine localization dynamics, which must be reconstructed by
comparing embryos fixed at different developmental stages. Consequently, in
most cases, the temporal sequence that leads to asymmetric localization in
wild type, or to lack of asymmetry in mutants, has not been determined. To
address this issue, we have developed a system to express and monitor GFP
fusions in live embryos (Reese et al.,
2000
; Strome et al.,
2001
). In this study, we have analyzed the localization dynamics
of PAR-2, PAR-6, MEX-5, MEX-6 and PIE-1 in wild type, and in embryos lacking
specific PAR or MEX activities. Our observations demonstrate that (1)
polarization involves distinct establishment and maintenance phases, (2) the
sperm asters function primarily by excluding the PAR-3/PAR-6/PKC-3 complex,
and (3) PAR-1 negatively regulates MEX-5/6 in the posterior.
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MATERIALS AND METHODS |
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JH1473 was constructed by crossing WH104/+ males with KK866 hermaphrodites
and selecting progeny that expressed both GFPs. WH104 was obtained by
bombarding pJH4.66 unc-119 inserted at a NaeI site into
unc-119 hermaphrodites (Strome et
al., 2001).
The GFP:PAR-2, GFP:PAR-6 and PIE-1:GFP transgenes were tested for rescue of corresponding mutants (data not shown). All rescued, indicating that the fusions are functional.
Time-lapse microscopy
GFP localization dynamics were analyzed by time-lapse microscopy: single
focal plane images, focused midway through the embryo (cross section), were
collected typically at 20 second intervals over an approx. 25-minute period
from pronuclear formation to the first cleavage. (Embryos that failed to
divide within 35 minutes were discarded). At each time point, both Nomarski
(0.01 seconds exposure) and fluorescence images (0.1 seconds exposure) were
collected. Images were acquired using a Photometrics CoolSnap FX digital
camera attached to a Zeiss Axioplan 2 equipped with Ludl shutters and a
mercury lamp. Acquisition scripts were written using IPLab software, and
acquired images were processed into QuickTime movies using 4D turnaround
software (Laboratory of Optical and Computational Imaging, University of
Wisconsin, Madison). Signal intensity cannot be compared between movies as the
range displayed is not constant. In all movies, the maternal pronucleus is to
the left and the paternal pronucleus is to the right.
par-1(RNAi)mex-5(RNAi)mex-6(RNAi) embryos (5/5) had an extra maternal
pronucleus significance is not known. Movies can be viewed at
ftp://www.wormbase.org/pub/wormbase/datasets/seydoux_2003.
RNA-mediated interference (RNAi)
RNAi was performed using the feeding method
(Timmons and Fire, 1998), and,
in the case of par-1, also by injection. Bacteria were grown
overnight on NNGM plates containing 60 µg/ml ampicillin and 80 µg/ml
IPTG. L4 hermaphrodites were allowed to feed for 24 hours at 25°C before
video microscopy. par-2, par-3, par-5, par-6, pkc-3 (RNAi) embryos
divided symmetrically; in contrast par-1(RNAi) embryos often divided
asymmetrically.
Immunolocalization
Immunostainings were performed as described previously
(Guo and Kemphues, 1995;
Etemad-Moghadam, 1995; Hung and Kemphues,
1999
; Tabuse et al.,
1998
).
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RESULTS |
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Localization dynamics in wild-type zygotes
Summary of zygote development
Immediately following fertilization, zygotes resume meiosis and begin
synthesizing an eggshell. Zygotes are fragile during this period; therefore we
typically began our time-lapse analysis after the completion of meiosis,
around the time that the maternal and paternal pronuclei first appear. Before
pronuclear formation, the cortex is very active and is undergoing intense
ruffling throughout the length of the embryo
(Fig. 1A). Following the
appearance of pronuclei, ruffling stops abruptly in a small area near the
sperm pronucleus. At that time, internal cytoplasm begins to flow towards the
sperm pronucleus and superficial cytoplasm flows away from it (this flow is
easily visualized by following the movement of individual yolk granules)
(Golden, 2000). As flow
proceeds, the smooth area in the posterior expands anteriorly
(Fig. 1B). Eventually ruffling
is confined to the anterior half of the embryo and culminates in a transient
but deep invagination of the membrane (pseudocleavage furrow;
Fig. 1C). After pseudocleavage,
ruffling ceases entirely, the pronuclei meet in the posterior
(Fig. 1D), migrate back towards
the center, rotate so that the duplicated centrosomes become aligned along the
AP axis (Fig. 1E), and undergo
nuclear membrane breakdown. The mitotic spindle forms initially in the center
of the embryo, but becomes displaced posteriorly during anaphase
(Fig. 1F), resulting in a
larger anterior cell (somatic blastomere AB) and a smaller posterior cell
(germline blastomere P1; Fig.
1G).
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GFP:PAR-2 and GFP:PAR-6
Before pronuclear formation, GFP:PAR-2 and GFP:PAR-6 were distributed
uniformly throughout the embryo. Both could be detected readily in the
cytoplasm, and were also visibly enriched at the cortex
(Fig. 1A, Movies 1 and 2).
The first asymmetry was seen after the appearance of pronuclei when ruffling ceased abruptly near the paternal pronucleus. At that time, GFP:PAR-2 began to increase and GFP:PAR-6 began to decrease on the cortex in the smooth region. For the next 10 minutes, as the smooth region expanded towards the anterior, the GFP:PAR-2 domain expanded along with it, whereas GFP:PAR-6 receded, becoming more prominent in the area where ruffling is maintained (Fig. 1B). By pseudocleavage (Fig. 1C), the fusions reached their final configurations on the cortex, with GFP:PAR-2 enriched in the posterior and GFP:PAR-6 enriched in the anterior. We also detected GFP:PAR-2 on centrosomes during pronuclear rotation (Movie 1) and GFP:PAR-6 in both pronuclei just before pronuclear fusion (Movie 2 and Fig. 1E). The latter localizations have not been reported previously for the endogenous proteins.
To examine the relationship between onset of polarity and formation of the sperm MTOC, we filmed embryos co-expressing GFP:PAR-2 and GFP:tubulin. In four out of the four embryos examined, we observed a good correlation, in timing and location, between appearance of the MTOC, local cessation of ruffling, and accumulation of PAR-2 on the cortex in the smooth zone. This correlation held even in embryos where the MTOC formed at a lateral position before moving to the pole. In these embryos, the GFP:PAR-2 domain initially also formed laterally, but rapidly shifted towards the pole along with the sperm pronucleus/MTOC complex (Movie 3, Fig. 2A-D).
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In six out of 11 embryos, we observed GFP:PAR-2 in a second domain on the
cortex next to the maternal pronucleus (Movie 4,
Fig. 2E-H). This domain
persisted until pseudocleavage and quickly disappeared afterwards. Transient
localization in the anterior cortex was also described for endogenous PAR-2 in
immunofluorescence experiments (Boyd et
al., 1996). The significance of this localization is not known,
but may be due to the transient influence of the meiotic spindle in this area
of the cortex (Wallenfang and Seydoux,
2000
).
GFP:MEX-5, GFP:MEX-6 and PIE-1:GFP
PIE-1:GFP and GFP:MEX-5 were initially uniformly distributed throughout the
cytoplasm and began to increase (PIE-1) or decrease (MEX-5) in the posterior
cytoplasm soon after the appearance of a smooth zone near the paternal
pronucleus (Fig. 3, Movies 5
and 6). The fusion proteins also appeared to decrease (PIE-1) or increase
(MEX-5) at the opposite end of the embryo. Maximal asymmetry was reached by
pseudocleavage with PIE-1:GFP enriched in the posterior and GFP:MEX-5 enriched
in the anterior (Fig. 3E). For
both fusions, localization was not absolute, with some GFP fluorescence
remaining in the opposite domain. Overall, the timing of PIE-1:GFP and
GFP:MEX-5 localization was similar to that of GFP:PAR-2 and GFP:PAR-6. Small
differences in timing cannot be excluded, however, since the fusions were not
visualized in the same embryos.
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GFP:MEX-6 (Movie 7) behaved essentially like GFP:MEX-5, consistent with the
fact that these proteins are 70% identical in sequence and function
redundantly (Schubert et al.,
2000). PIE-1:GFP, GFP:MEX-5 and GFP:MEX-6 all accumulated on
granules in germline blastomeres. Localization on P granules has been reported
for PIE-1 and MEX-5 (Mello et al.,
1996
; Schubert et al.,
2000
). Since antibodies specific to MEX-6 are not yet available,
we do not know whether the GFP:MEX-6 pattern matches that of endogenous
MEX-6.
Polarization of the zygote involves two distinct phases
To test whether the sperm asters function by recruiting PAR-2 or by
excluding the PAR-3/PAR-6/PKC-3 complex from the posterior, we examined the
distribution of GFP:PAR-2 and GFP:PAR-6 in embryos where par-2, par-3,
par-6 or pkc-3 were inactivated by RNA-mediated interference
(RNAi) (Fire et al., 1998).
In par-3(RNAi), pkc-3(RNAi) and par-6(RNAi),
(n=3 time lapses for each), high levels of GFP:PAR-2 were observed
throughout the cortex from pronuclear formation to the first cleavage
(Fig. 1 and Movies 8-10),
consistent with previous immunofluorescence observations of par-3 and
par-6 mutants (Boyd et al.,
1996; Watts et al.,
1996
). Occasionally stronger patches of GFP:PAR-2 fluorescence
appeared transiently and unpredictably at one of the two poles. We conclude
that, in the absence of the anterior PAR complex, PAR-2 can accumulate on the
cortex but cannot become asymmetric.
In par-2(RNAi) embryos (n=21), GFP:PAR-6 started out uniformly distributed at the cortex (Fig. 1 and Movie 11). As in wild type, when ruffling stopped in the posterior, GFP:PAR-6 disappeared from that area, eventually receding to 63% egg length (average from 11 time-lapse examinations). After pronuclear meeting, however, GFP:PAR-6 crept back towards the posterior (Fig. 1E). By cytokinesis (Fig. 1F), GFP:PAR-6 was found throughout the cortex. This behavior (initial asymmetry followed by loss of asymmetry) was observed in 2 independent GFP:PAR-6 lines (total of 21 time-lapse examinations).
Because this behavior had not been noted in previous immunofluorescence
studies (Hung and Kemphues,
1999) and to exclude the possibility that it was due to an
artifact of RNAi or the GFP fusion, we reexamined the distribution of
endogenous PAR-3, PAR-6 and PKC-3 in par-2(0) mutants by
immunofluorescence using DAPI staining to stage embryos
(Table 2). Those data confirmed
the GFP:PAR-6 results. We conclude that PAR-3, PAR-6 and PKC-3 can accumulate
at the cortex and become enriched in the anterior in the absence of PAR-2.
However, they require PAR-2 to remain excluded from the posterior after
pronuclear meeting. These data indicate that polarization of the cortex
involves two phases: a PAR-2-independent "establishment" phase
before pronuclear meeting, and a PAR-2-dependent "maintenance"
phase afterwards.
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In par-3(RNAi) (n=7) and pkc-3(RNAi)
(n=8) embryos, GFP:PAR-6 failed to accumulate on the cortex from the
earliest stage examined (pronuclear formation; Movies 12-13). This result is
consistent with previous studies, which indicated that PAR-3, PAR-6 and PKC-3
depend on each other for cortical localization
(Watts et al., 1996;
Hung and Kemphues, 1999
;
Tabuse et al., 1998
). We
observed, however, transient accumulation of GFP:PAR-6 in pronuclei just
before mitosis, as is observed in wild type. We conclude that par-3
and pkc-3 are essential for cortical localization of PAR-6 throughout
the first cell cycle, but are dispensable for PAR-6's transient nuclear
localization.
The non-muscle myosin NMY-2 and the 14-3-3 protein PAR-5 are required
during the establishment phase
NMY-2
NMY-2 is required for embryonic polarity and PAR localization
(Guo and Kemphues, 1996). To
determine whether NMY-2 is required for the establishment or maintenance
phases, we examined GFP:PAR-2 and GFP:PAR-6 in nmy-2(RNAi) embryos
(Materials and Methods). nmy-2(RNAi) embryos fall into three classes,
depending on the severity of loss of nmy-2 activity, from strong to
weak (Guo and Kemphues,
1996
).
Class I. Embryos in this class do not undergo ruffling, pseudocleavage or
cytokinesis. Pronuclei form and migrate normally in these embryos, eventually
resulting in the formation of a symmetric spindle
(Guo and Kemphues, 1996).
These phenotypes are consistent with embryos lacking actin contractility but
retaining normal microtubules. In one embryo examined of this class, GFP:PAR-6
remained uniformly distributed at the cortex throughout the first cell cycle
(n=1, Movie 14). GFP:PAR-2 did not become visibly enriched on the
cortex (n=3), and instead was maintained throughout the cytoplasm and
also on foci around centrosomes (Fig.
4; Movie 15). The lack of cortical PAR-2 in nmy-2(RNAi)
embryos was dependent on PAR-6: nmy-2(RNAi);par-6(RNAi) embryos
exhibited strong GFP:PAR-2 at the cortex (n=3;
Fig. 4; Movie 16). We conclude
that NMY-2 is required for onset of the establishment phase and that, in its
absence, the PAR-3/PAR-6/PKC-3 complex occupies the entire cortex, excluding
PAR-2.
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Class II and Class III. These embryos developed cytokinesis furrows that
were either transient (Class II) or lead to the formation of two equal size
cells (Class III). Like Class I embryos, these embryos maintained GFP:PAR-6
uniformly at the cortex throughout the first cell cycle (n=2,
Fig. 4, Movie 17). Unlike Class
I embryos, however, Class II and Class III embryos developed one or two
patches of GFP:PAR-2 at the cortex by pronuclear meeting (n=6,
Fig. 3, Movies 18, 19). These
patches were similar to those seen for endogenous PAR-2 in fixed
nmy-2(RNAi) and mlc-4(RNAi) embryos
(Guo and Kemphues, 1996;
Shelton et al., 1999
). Our
time-lapse analysis indicates that these patches form later than in wild type
(pronuclear meeting rather than pronuclear formation) and occur only in
embryos with residual actin contractility (as evidenced by ingressing
furrows). We conclude that the PAR-2 patches observed in nmy-2(RNAi)
embryos are not the result of an early, myosin-independent step during the
establishment phase, but rather correspond to a later response of PAR-2 that
is still dependent on NMY-2.
In all nmy-2(RNAi) embryos (n=8), we also detected
GFP:PAR-2 in foci around the pronuclei or spindle, as was reported previously
for mutants that fail to polarize normally
(Rappleye et al., 2002). In
four out of eight time-lapse analyses, GFP:PAR-2 appeared to shuttle from the
cortex to the nearest pronucleus or centrosome. The significance of this
behavior is not known but suggests that PAR-2 may have an affinity for
microtubules.
PAR-5
par-5 mutant embryos exhibit extensive overlap between PAR-3 and
PAR-2 and between PAR-1 and PKC-3 (Morton
et al., 2002), indicating that PAR-5 is essential for creating
and/or maintaining distinct anterior and posterior PAR domains. To determine
whether PAR-5 functions during the establishment phase or the maintenance
phase, we examined GFP:PAR-2 and GFP:PAR-6 dynamics in par-5(RNAi)
embryos.
We found that par-5(RNAi) embryos maintain GFP:PAR-6 (n=9) and GFP:PAR-2 (n=5) at the cortex from pronuclear formation through cleavage (Fig. 1, Movies 20-21). We observed a reproducible reduction in GFP:PAR-6 fluorescence in the posterior when ruffling ceased in that area. This reduction, however, appeared later and was not as pronounced as in wild type, with detectable levels of GFP:PAR-6 remaining in the region. After pseudocleavage, GFP:PAR-6 reappeared uniformly throughout all the cortex. During the period that GFP:PAR-6 decreased in the smooth zone, we did not observe a reproducible increase in GFP:PAR-2. Occasionally we observed patches of stronger or lower GFP:PAR-2 fluorescence in certain areas of the cortex, but these patches were transient and did not appear in a predictable pattern.
We conclude that PAR-5 during the establishment phase is required (1) for maximal response of the PAR-3/PAR-6/PKC-3 to the sperm aster cue and (2) to prevent overlap between PAR-3/PAR-6/PKC-3 and PAR-2.
PAR-1 inhibits and MEX-5 and MEX-6 promote, expansion of the
posterior domain during the establishment phase
Mutations in par-1 have been reported not to affect the
localization of other PAR proteins in zygotes
(Boyd et al., 1996;
Etemad-Moghadam et al., 1995
;
Hung and Kemphues, 1999
).
Consistent with this, we found that GFP:PAR-2 was asymmetric in
par-1(RNAi) embryos, in clear contrast to what we observed in embryos
lacking par-3, par-6, pkc-3 or par-5. We noted, however,
that the anterior-most boundary of the PAR-2 domain was shifted towards the
anterior in par-1(RNAi) zygotes (n=6;
Fig. 5B; Movie 22). This
displacement coincided spatially and temporally with the anteriorly displaced
pseudocleavage of par-1 embryos
(Kirby et al., 1990
). To
confirm that this effect was not an artifact of RNAi or the GFP:PAR-2 fusion,
we examined par-1(b274) embryos for endogenous PAR-2 by
immunofluorescent staining. We observed a similar anterior expansion of the
PAR-2 domain in embryos at pronuclei meeting (6/6; data not shown).
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PAR-1 regulates the localization of cytoplasmic proteins, including MEX-5
and PIE-1 (Schubert et al.,
2000; Tenenhaus et al.,
1998
). We confirmed these observations and extended them to MEX-6,
by following GFP:MEX-5, GFP:MEX-6 and PIE-1:GFP dynamics in
par-1(RNAi) embryos. In all cases, the fusions remained uniformly
distributed (Movies 23-25). To determine whether expansion of the PAR-2 domain
in par-1 zygotes was due to ectopic MEX-5/6 and/or PIE-1, we examined
GFP:PAR-2 dynamics in par-1(RNAi) mex-5(RNAi) mex-6(RNAi) and
par-1(RNAi) pie-1(RNAi) zygotes (Movies 26-27). We found that
expansion of the PAR-2 domain was suppressed in par-1(RNAi) mex-5(RNAi)
mex-6(RNAi) embryos (n=5;
Fig. 5N), but not in
par-1(RNAi) pie-1(RNAi) embryos (n=3;
Fig. 5M). Suppression of PAR-2
expansion was also observed in par-1(it51) mex-5(RNAi) mex-6(RNAi)
embryos stained for endogenous PAR-2 (5/5; data not shown). These results
indicate that expansion of the PAR-2 domain in par-1 zygotes is due
to ectopic MEX-5 and MEX-6.
To explore further the role of MEX-5/6, we examined PAR-2 dynamics in
embryos depleted of these two proteins by RNAi. mex-5;mex-6 double
mutants were reported not to affect PAR asymmetry
(Schubert et al., 2000).
Consistent with those results, we found that GFP:PAR-2 was asymmetric in most
mex-5(RNAi) mex-6(RNAi) zygotes (5/7). However, GFP:PAR-2 dynamics
were clearly aberrant in those embryos. In wild type, the GFP:PAR-2 domain
expands quickly and reaches its maximal domain (close to 50% egg length) by
pseudocleavage (Fig. 1). In
contrast, in five out of seven mex-5(RNAi)mex-6(RNAi) embryos, the
GFP:PAR-2 domain remained small (33% egg length) throughout pronuclear
migration and began to expand only after pronuclear meeting, eventually
reaching its normal distribution just before the first cleavage
(Fig. 5G-L, Movie 28). This
pattern was essentially opposite that seen in par-1(RNAi) embryos,
where the PAR-2 domain extends further than normal
(Fig. 5A-F). In the other 2
embryos examined (2/7), PAR-2:GFP was never seen at the cortex (Movie 29).
Unlike most mex-5(RNAi);mex-6(RNAi) embryos, which divide
asymmetrically, these two embryos divided symmetrically. These defects were
specific to mex-5 and mex-6, as pie-1(RNAi) embryos
exhibited normal GFP:PAR-2 dynamics and divided asymmetrically (n=3,
Movie 30).
To verify that the mex-5/6 results were not an artifact of RNAi or GFP fusions, we re-examined the distribution of endogenous PAR-1 and PAR-2 in mex-5(zu199)mex-6(pk440) double mutants by immunofluorescence, using DAPI staining to stage embryos. This analysis confirmed that establishment of the PAR-1/PAR-2 domain is delayed in many mex-5(-)mex-6(-) zygotes, and occasionally does not occur at all [Fig. 5P; 18/27 zygotes had PAR-1 domains smaller than wild type; similar results were also obtained for PAR-2 (not shown)]. Examination of two-cell mex-5(zu199)mex-6(pk440) double mutants confirmed that these embryos occasionally undergo a symmetric first cleavage (19/133).
We conclude that MEX-5 and MEX-6 are required to promote rapid and consistent expansion of the posterior domain during the establishment phase. MEX-5/6 could affect growth of the posterior domain directly by promoting PAR-1/PAR-2 localization there, or indirectly, by excluding the anterior PARs from that region. To distinguish between these possibilities, we analyzed GFP:PAR-6 dynamics in par-1(RNAi) and mex-5(RNAi);mex-6(RNAi) embryos. We found that five out of six par-1 embryos had a smaller GFP:PAR-6 domain at pseudocleavage (Movie 31), and one out of mex-5(RNAi);mex-6(RNAi) embryos maintained uniform PAR-6:GFP throughout the first cell cycle (Movie 32). Similarly, 11 out of 19 mex-5(zu199)mex-6(pk440) had expanded PAR-3 domains (Fig. 5R). These phenotypes are unlikely to be a secondary consequence of the smaller PAR-2 domain observed in these embryos, since par-2 is not essential to exclude anterior PARs from the posterior during the establishment phase (Fig. 1). We conclude that MEX-5/6 regulate expansion of the posterior domain by helping to exclude anterior PARs from that region.
In the course of analyzing GFP:PAR-6 dynamics in par-1(RNAi)
embryos, we noticed that PAR-6 asymmetry became significantly less pronounced
after pronuclear meeting (Movie 31). Whereas par-2(RNAi) embryos lose
all GFP:PAR-6 asymmetry after pronuclear meeting, par-1(RNAi) embryos
appeared to regain asymmetric GFP:PAR-6 at the two-cell stage. This is
consistent with previous analyses, which demonstrated that par-1
mutants maintain anteriorly enriched PAR-3 and PAR-6 even after undergoing a
symmetric cleavage (Boyd et al.,
1996; Etemad-Moghadam et al.,
1995
). We conclude that PAR-1 contributes to PAR asymmetry during
the maintenance phase, although unlike PAR-2, it may not be essential after
cleavage.
PAR-1 inhibits MEX-5/6 activity and/or levels
The opposite phenotypes of par-1 and mex-5/6 mutants
during the establishment phase, and the fact that mex-5/6 are
epistatic to par-1 (Fig.
5) indicate that PAR-1 negatively regulates MEX-5/6. Two models
could account for this negative regulation. PAR-1 could inhibit MEX-5/6 by
causing them to relocate to the anterior, or PAR-1 could inhibit MEX-5/6 by
negatively regulating their activity or level in the posterior. The latter
predicts that mutants with uniform PAR-1 would have low MEX-5/6 activity
and/or levels throughout the zygote. We have obtained evidence in support of
this prediction, using PIE-1:GFP to monitor MEX-5/6 activity.
MEX-5 and MEX-6 are required for PIE-1 asymmetry
(Schubert et al., 2000). As
expected, PIE-1:GFP remained uniform in mex-5(RNAi)mex-6(RNAi)
embryos (n=4; Movie 33). [In contrast, GFP:MEX-5 localized normally
in pie-1(RNAi) embryos (n=3; Movie 34)]. During these
analyses, we noticed that the cytoplasmic:nuclear ratio of PIE-1 is also
regulated by MEX-5/6. In wild-type embryos, during pronuclear migration,
PIE-1:GFP accumulated progressively with time in the sperm pronucleus
(posterior), but not in the maternal nucleus (anterior,
Fig. 3D). After cleavage,
PIE-1:GFP accumulated in the P1 nucleus (posterior) but remained excluded from
the AB nucleus (anterior, Fig.
3H). This difference is dependent on MEX-5/6: PIE-1:GFP was found
in both pronuclei in mex-5(RNAi)mex-6(RNAi) zygotes (n=4,
scored at pronuclear meeting stage), and in neither pronucleus in
par-1(ax54) zygotes (n=6), where MEX-5 and MEX-6 remain
uniformly distributed (Fig. 6). We confirmed that PIE-1's low nuclear:cytoplasmic ratio in par-1
mutants was dependent on MEX-5/6, by removing MEX-5/6 by RNAi in
par-1(ax54) embryos. As expected, we detected PIE-1:GFP in both
pronuclei in par-1(ax54)mex-5(RNAi)mex-6(RNAi) zygotes (n=8;
Fig. 6).
|
In par-3 embryos, PAR-1 is uniformly distributed throughout the
cortex (Guo and Kemphues, 1995)
and MEX-5 (and presumably MEX-6) remains uniformly distributed throughout the
cytoplasm (Fig. 6) (see also
Schubert et al., 2000
).
Strikingly, we found that PIE-1:GFP accumulates in both pronuclei in
par-3(it71) zygotes (n=4), as is seen in
mex-5(RNAi)mex-6(RNAi) embryos
(Fig. 6). This observation
indicated that MEX-5/6 is not active, or does not accumulate to a critical
threshold level, in par-3 zygotes. A likely explanation for this
result is that uniformly distributed cortical PAR-1 reduces MEX-5/6 activity
or level equally throughout the cytoplasm. If so, removal of PAR-1 in
par-3 mutants should restore MEX-5/6 activity, or accumulation, and
keep PIE-1 out of the pronuclei. In agreement with this prediction,
par-3(it71);par-1(RNAi) double mutants exhibited the same low PIE-1
nuclear:cytoplasmic ratio that is observed in par-1 mutants
(n=10; Fig. 6). We
conclude that PAR-1 can inhibit MEX-5/6 activity even under conditions where
it does not create MEX-5/6 asymmetry. These observations suggest that PAR-1
acts on MEX-5/6 by negatively regulating their activity, level, or both.
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DISCUSSION |
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Establishment
Previous studies have implicated the sperm-derived MTOC as the most likely
source for the spatial cue that initially polarizes the zygote
(Sadler and Shakes, 2000;
O'Connell et al., 2000
;
Wallenfang and Seydoux, 2000
).
Our time-lapse analysis supports this view. Formation of the MTOC correlates
temporally and spatially with the earliest evidences of polarity: (1)
cessation of ruffling, (2) enrichment of GFP:PAR-2, and (3) loss of GFP:PAR-6
in the posterior cortex. Our data also demonstrates that the primary effect of
the polarizing cue is to clear the PAR-3/PAR-6/PKC-3 complex from the
posterior cortex. This effect does not require PAR-2. In contrast, restriction
of PAR-2 to the posterior requires PAR-6, PAR-3 and PKC-3, suggesting that
PAR-2 does not sense the polarity cue directly but instead responds to local
displacement of the anterior complex.
The establishment phase requires the class II non-muscle myosin, NMY-2:
nmy-2(RNAi) prevents PAR-6 (and presumably associated PAR-3 and
PKC-3) from sensing the polarity cue, causing it to remain uniformly
distributed throughout the cortex. In NMY-2-depleted embryos, PAR-2 is
prevented from accumulating at the cortex by PAR-6 (and/or its partners). This
`default' state of PAR-6 on/PAR-2 off is also observed in mutants lacking
sperm asters (O'Connell et al.,
2000; Wallenfang and Seydoux,
2000
) and in mutants where the MTOC detaches from the cortex
prematurely (Rappleye et al.,
2002
). These observations suggest that the initial
symmetry-breaking event involves signaling between the MTOC and the actin
cytoskeleton. Consistent with this view, one of the earliest signs of
polarization is cessation of ruffling in the cortex nearest the MTOC.
Cessation of ruffling correlates with MTOC formation, but does not appear to
require PAR activity (cessation of ruffling was observed in all par
mutants examined in this study). These observations suggest that modification
of the actin cytoskeleton may be an obligatory step before the onset of PAR
asymmetry. We propose that signaling from the MTOC modifies the actin
cytoskeleton locally, which causes the PAR-3/PAR-6/PKC-3 complex to become
destabilized, allowing PAR-2 to accumulate in its place.
The establishment phase also requires the 14-3-3 protein PAR-5. In its
absence, PAR-6 responds only weakly, if at all, to the polarity cue and PAR-2
is no longer excluded from the cortex by the PAR-3/PAR-6/PKC-3 complex.
Cessation of ruffling in the posterior, however, still occurs in
par-5(RNAi) embryos, suggesting that PAR-5 is not required for the
initial MTOC/actin cytoskeleton interaction. Although this interpretation is
complicated by the fact that residual PAR-5 activity may persist in
par-5(RNAi) embryos (Morton et
al., 2002), we propose that PAR-5 functions primarily by
regulating the ability of the PAR-3/PAR-6/PKC-3 complex to (1) exclude PAR-2
and (2) respond to changes in the cytoskeleton. The presence of a potential
14-3-3 binding motif in PAR-3 (Morton et
al., 2002
) is consistent with the possibility that PAR-5 regulates
the PAR-3/PAR-6/PKC-3 complex by binding to it directly.
A feedback loop during the establishment phase
Surprisingly, we found that the predominantly cytoplasmic MEX-5 and MEX-6
also play a role during the establishment phase. In the absence of MEX-5 and
MEX-6, the posterior domain occasionally does not form (15-30% of embryos),
and frequently (50% or more of embryos) is slow to reach its final
configuration. These observations indicate that, although MEX-5 and MEX-6 are
not absolutely required for PAR localization in the zygote
(Schubert et al., 2000), they
do play a role in ensuring a robust response by the PAR-3/PAR-6/PKC-3 complex
to the MTOC/actin cytoskeleton signal.
We have found that this aspect of MEX-5/6 function is negatively regulated by PAR-1. In par-1 mutants, MEX-5 and MEX-6 cause the posterior domain to extend further towards the anterior during the establishment phase. Since PAR-1 itself becomes enriched in the posterior domain, one attractive possibility is that PAR-1 and MEX-5/6 participate in a feedback loop that limits expansion of the posterior domain. We propose the following model. At the beginning of the establishment phase, MEX-5 and MEX-6 levels are high throughout the zygote and help clear the PAR-3/PAR-6/PKC-3 complex from the region nearest the sperm asters. This clearing allows PAR-2 and PAR-1 to accumulate on the cortex, which in turn reduces MEX-5/6 activity and/or levels in the surrounding cytoplasm. Eventually, MEX-5/6 levels become too low to fuel further expansion of the posterior domain. We do not yet know whether the partial penetrance of the mex-5();mex-6() phenotype is due to redundancy with other factors, or is indicative of a minor role for the feedback loop in regulating PAR asymmetry.
Maintenance
The finding that the sperm-derived MTOCs play a role in initiating polarity
raised the question of how polarity is maintained after pronuclear meeting,
when the pronuclei/centrosome complex rotates and microtubules invade the
anterior end of the embryo (Fig.
7). Our observations provide an answer: PAR-2. We have found that
in the absence of PAR-2, PAR-3 and PAR-6 and PKC-3 can become asymmetric
before pronuclear meeting, but return into the posterior domain afterwards.
This finding demonstrates two points: (1) the PAR-6/PAR-3/PKC-3 complex no
longer responds to the MTOC-dependent cue after pronuclear meeting, and (2)
PAR-2 is required after pronuclear meeting, but not earlier, to exclude the
PAR-6/PAR-3/PKC-3 complex from the posterior. We propose that pronuclear
meeting (and/or the end of prophase) triggers a change in the cytoskeleton, or
in the PAR-6/PAR-3/PKC-3 complex, which turns off the MTOC-dependent polarity
signal, or the ability to respond to it. From that point on, PAR-2 becomes
essential to keep PAR-6/PAR-3/PKC-3 out of the posterior cortex. It is
intriguing that PAR-6 briefly localizes to nuclei at pronuclear meeting,
raising the possibility that it becomes modified at that time.
The existence of distinct establishment and maintenance phases is also
supported by the observation that cdc-42 is required after prophase,
but not earlier, for PAR-3, PAR-6 and PKC-3 asymmetry
(Gotta and Ahringer, 2001). Our
analysis of GFP:PAR-6 dynamics in par-1(RNAi) embryos suggests that
PAR-1 also contributes to maintenance of PAR asymmetry after pronuclear
meeting. How PAR-2, CDC-42 and PAR-1 function together to maintain the balance
between anterior and posterior PAR domains remains to be determined.
Asymmetric protein localization in C. elegans zygotes
During the establishment phase, MEX-5, MEX-6 and PIE-1 asymmetries appear
in the cytoplasm with approximately the same temporal dynamics as PAR
asymmetries on the cortex. In agreement with previous studies, we have found
that anterior localization of MEX-5 and MEX-6 is dependent on PAR-1, and
posterior localization of PIE-1 is dependent on PAR-1 and on MEX-5/6. Since
PAR-1 is not essential for asymmetric localization of other PARs during the
establishment phase, these findings are consistent with PAR-1 being the PAR
protein most directly required for localization of cytoplasmic factors.
PAR-1 could localize MEX-5 and MEX-6 by promoting their translocation to the anterior or by negatively regulating their activity and/or stability in the posterior. We have obtained evidence in support of the latter by analyzing the effect of delocalized PAR-1 on MEX-5/6 activity. In these experiments, we used the nuclear:cytoplasmic ratio of PIE-1:GFP as a readout for MEX-5/6 activity. We found that MEX-5/6 activity is low in par-3() zygotes where PAR-1 is delocalized. This low activity is dependent on PAR-1: zygotes lacking both par-3 and par-1 have high MEX-5/6 activity (Fig. 6). We conclude that PAR-1 can inhibit MEX-5/6 activity even under conditions where MEX-5/6 do not become asymmetric. We do not know yet whether this inhibition depends on lowering MEX-5/6 levels, inhibiting their activity, or a combination of both, as attempts to quantify protein levels in vivo have been unsuccessful thus far.
These results suggest that restriction of PAR-1 to the posterior may be
necessary to generate cytoplasmic asymmetries, but conflict with previous
evidence. In par-2 mutant embryos, PAR-1 is not detectably cortical
or asymmetric (Boyd et al.,
1996), yet P granules (Boyd et
al., 1996
) and PIE-1
(Tenenhaus et al., 1998
)
become asymmetric in the zygote. One possibility is that asymmetric activation
of PAR-1 still occurs in these mutants in the absence of localization. Future
experiments monitoring PAR-1 dynamics in live embryos will be needed to
clarify the roles of PAR-2 and PAR-1 in regulating cytoplasmic
asymmetries.
In addition to negatively regulating MEX-5/6 in the posterior, PAR-1 also
causes MEX-5 and MEX-6 to accumulate in the anterior. We do not yet know the
mechanism that mediates this enrichment. A possibility, which is consistent
with our data, is that local action of PAR-1 destabilizes MEX-5/6 in the
posterior, causing them to accumulate only in the anterior (owing to on-going
translation of these proteins from maternal RNA). Accumulation of MEX-5 and
MEX-6 in the anterior would in turn allow PIE-1 and other germline factors to
accumulate only in the posterior. This outcome is reminiscent of the effect of
the MTOC/actin cytoskeleton signal, which destabilizes or displaces the
PAR-6/PAR-3/PKC-3 complex from the posterior cortex, causing it to accumulate
only in the anterior, which in turn allows PAR-2 and PAR-1 to accumulate in
the posterior. An attractive possibility is that local changes in stability
among competing proteins is the dominant mechanism mediating asymmetric
localization in C. elegans zygotes. The finding that the
localizations of PAR-2, PAR-6, MEX-5, MEX-6 and PIE-1 do not require sequences
in untranslated regions (this study)
(Reese et al., 2000) already
suggests that mechanisms acting at the protein, rather than the RNA, level
prevail in the zygote. A challenge for the future will be to determine whether
these mechanisms regulate protein stability, movement, or both.
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ACKNOWLEDGMENTS |
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