1 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle,
WA 98109, USA
2 Center for Cell Dynamics and Friday Harbor Labs, Friday Harbor, WA 98250,
USA
Author for correspondence (e-mail:
jpriess{at}fhcrc.org)
Accepted 23 July 2003
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SUMMARY |
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Key words: C. elegans, Apicobasal, PAR-3, PAR-6, NMY-2, Nonmuscle myosin, Gastrulation, Ingression, Cell adhesion
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Introduction |
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Although the PAR proteins have been studied primarily in newly fertilized,
1-cell embryos, they are expressed continuously during the early cell cycles
and subsequently in epithelial cells during organogenesis. After the 1-cell
stage, the anterior-posterior asymmetry of the PAR proteins is reiterated only
in the lineage of cells that eventually produce the germline (germline
precursors; see Fig. 1A).
PAR-3, PAR-6 and PKC-3 associate with the anterior surface of each germline
precursor prior to division, and PAR-1 and PAR-2 associate with the posterior
surface (Boyd et al., 1996;
Etemad-Moghadam et al., 1995
;
Guo and Kemphues, 1995
;
Hung and Kemphues, 1999
;
Tabuse et al., 1998
). By
contrast, the embryonic cells that produce only somatic cell types (somatic
precursors) undergo a dramatic reorganization of PAR proteins
(Boyd et al., 1996
;
Etemad-Moghadam et al., 1995
;
Guo and Kemphues, 1995
;
Hung and Kemphues, 1999
;
Nance and Priess, 2002
;
Tabuse et al., 1998
). At the
early 4-cell stage, the formerly anterior PAR proteins, PAR-3, PAR-6 and
PKC-3, localize transiently to the entire cell cortex. By the late 4-cell
stage, however, PAR-3, PAR-6 and PKC-3 redistribute to the contact-free,
apical, surfaces of cells. The formerly posterior PAR proteins, PAR-1 and
PAR-2, localize in a reciprocal manner to sites of cell contact, the
basolateral surfaces. The PAR proteins gradually disappear from embryos after
the 26-cell stage. PAR-3, PAR-6 and PKC-3 are expressed again at the
400-cell stage in the epithelial cells of developing organs, where they
are localized asymmetrically toward the apical surface
(Leung et al., 1999
;
McMahon et al., 2001
). The
epithelial functions of the PAR proteins have not been studied in C.
elegans. However, Drosophila homologs of PAR-3, PAR-6 and PKC-3
are expressed in epithelial cells where they appear to distinguish apical from
nonapical membrane domains (reviewed by
Knust and Bossinger, 2003
).
Mutations in the Drosophila par homologs result in gross defects in
the polarity of epithelial cells and can cause epithelial cell sheets to
become multilayered (Müller and
Wieschaus, 1996
; Petronczki
and Knoblich, 2001
; Wodarz et
al., 2000
).
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Previous studies have shown that the pattern of cell contacts in the early
embryo determines the basal position of the blastocoel; 4-cell embryos that
are combined head-to-head along their former apical surfaces generate an
ectopic blastocoel between these surfaces
(Nance and Priess, 2002).
Interestingly, the PAR proteins have been shown to redistribute in these same
experiments, with PAR-3 and PAR-6 moving to the new contact-free surfaces.
Abnormal separations can develop between the lateral surfaces of cells in
par-3 and par-6 mutants, but not par-2 mutants, and
cell ingressions are either absent or abnormal
(Nance and Priess, 2002
)
(J.N., E.M.M. and J.R.P., unpublished). Thus, PAR-3 and PAR-6 might function
in apicobasal asymmetry of early embryonic cells.
Determining whether PAR-3 and PAR-6 function in apicobasal asymmetry in the
early embryo is complicated by their roles in anterior-posterior asymmetry at
the 1-cell stage. For example, par-3 mutant embryos have highly
abnormal patterns of cell cleavage and altered cell fates that might disrupt
apicobasal asymmetries indirectly
(Kemphues et al., 1988). PAR-3
and PAR-6 are encoded by maternally supplied mRNAs
(Kemphues et al., 1988
;
Watts et al., 1996
), and the
transition from anterior-posterior to apicobasal PAR asymmetry occurs without
embryonic gene transcription (Nance and
Priess, 2002
). Thus, preventing PAR-3 and PAR-6 function after the
1-cell stage requires a method that selectively removes maternally supplied
gene products.
In normal embryonic development, the maternal protein PIE-1 is distributed
asymmetrically to the germline precursors, in part through the degradation of
PIE-1 in somatic precursors (see Fig.
1A) (Mello et al.,
1996; Reese et al.,
2000
). Analysis of PIE-1 has identified a peptide sequence, the
ZF1 domain, which is necessary and sufficient for the degradation of PIE-1 in
somatic precursors (Reese et al.,
2000
). Because the anterior-posterior asymmetry of the PAR
proteins is established before the asymmetric degradation of PIE-1
(Tenenhaus et al., 1998
), we
reasoned that a PAR protein coupled to ZF1 might function in
anterior-posterior asymmetry before being degraded. We show here that ZF1
coupled to PAR-3 and PAR-6 proteins rescue all the anterior-posterior defects
associated with mutations in par-3 and par-6, respectively.
However, the coupled proteins disappear prior to gastrulation and the
resulting embryos have defects in lateral adhesion and cell ingression. Thus,
PAR-3 and PAR-6 have a role in apicobasal asymmetry in the early embryo that
is independent of their earlier role in anterior-posterior asymmetry.
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Materials and methods |
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par-3(ZF1) strains were lon-1 par-3; par-3::zf1::gfp(zuIs20);
him-8. par-6(ZF1) strains were unc-101 par-6;
par-6::zf1::gfp(zuIs54); other alleles of par-6::zf1::gfp were
used where indicated. Because par-3(ZF1) and par-6(ZF1)
strains were marked with lon-1 and unc-101 mutations,
respectively, lon-1 and unc-101 worms were used as controls
for these strains. The him-8 mutation present in the
par-3(ZF1) strain increases the frequency of males and does not alter
early embryogenesis (Hodgkin et al.,
1979) (J.N., E.M.M. and J.R.P., unpublished). A strain of genotype
unc-32 par-3; par-3::zf1::gfp(zuIs20) was used to assess the
viability of par-3(ZF1) embryos.
Plasmid construction
Standard techniques were used to manipulate and amplify DNA. Genomic
sequences containing par-3, par-6 and nmy-2 were identified
using the Wormbase web site
(http://www.wormbase.org,
release WS54, 2001). Cosmid DNA containing each gene was digested
(par-3: F54E7, 16526 bp SalI fragment; par-6:
T26E3, 9080 bp XbaI-SmaI fragment; nmy-2: F20G4,
13965 bp XbaI fragment) and subcloned into the pBluescript KS+ vector
(Stratagene). Subclones of each gene are predicted to include the entire
coding region, 3'-untranslated region and 3-5 kb of sequence 5' of
the gene. A PstI site was introduced either just before or in the
stop codon of each gene by site-directed mutagenesis (Quickchange kit,
Stratagene) or recombinant PCR. To construct par-6::gfp and
nmy-2::gfp, the coding region of gfp was amplified by PCR
from plasmid pPD95.75 (1995 Fire Lab vector kit,
www.ciwemb.edu)
and cloned into the introduced PstI site. To construct
par-3::zf1::gfp and par-6::zf1::gfp, sequences encoding the
ZF1 domain (Reese et al.,
2000) were first amplified by PCR from pie-1 cDNA p661
(Mello et al., 1996
) and
introduced into the KpnI site 5' of gfp in plasmid
pPD95.75; the resulting zf1::gfp coding region was amplified by PCR
and cloned into the introduced PstI site. Prior to transformation of
each construct, the unc-119(+) coding region was inserted into the
NotI site in the vector (Maduro
and Pilgrim, 1995
).
To construct end-1::gfp, the end-1 promoter (2126 bp
5' to 75 bp 3' of the end-1 start codon) was amplified by
PCR and fused 5' of gfp coding sequences in plasmid pPD95.75
(Cassata et al., 1998).
Worm transformations
Strains expressing par-3::zf1::gfp, par-6::gfp, par-6::zf1::gfp
and nmy-2::gfp were obtained by microparticle bombardment of
unc-119 worms with plasmids described above
(Praitis et al., 2001). A
strain expressing the end-1::gfp reporter was obtained by injecting
spe-26 worms with end-1::gfp and a spe-26(+)
cotransformation marker; the resulting end-1::gfp extrachromosomal
array was integrated by
-irradiation
(Mello and Fire, 1995
).
Antibodies and immunostaining
Anti-PAR-3 monoclonal antibody P4A1 was produced in collaboration with Ken
Kemphues. Purified recombinant PAR-3
(Etemad-Moghadam et al., 1995)
was injected into mice at the FHCRC Hybridoma Production Facility as described
(Wayner and Carter, 1987
).
Hybridoma supernatants were assayed by immunostaining early embryos fixed in
bulk with paraformaldehyde and methanol
(Costa et al., 1997
). Antibody
P4A1 stained early embryos in the same pattern as previously described PAR-3
polyclonal sera (Etemad-Moghadam et al.,
1995
) and did not stain either par-3(it71) or
par-3(RNAi) early embryos (data not shown).
For most immunostaining experiments, embryos were fixed on slides using the
freeze-crack methanol procedure and incubated with primary antibodies and
fluorochrome-conjugated secondary antibodies
(Leung et al., 1999); embryos
were fixed for PIE-1 immunostaining as described
(Mello et al., 1996
). The
following primary antibodies/antisera and dilutions were used: chicken
anti-GFP, 1:200 (Chemicon); mouse anti-HMP-1, 1:10
(Costa et al., 1998
); rabbit
anti-HMR-1, 1:10 (Costa et al.,
1998
); rabbit anti-LAD-1, 1:300
(Chen et al., 2001
); rabbit
anti-PAR-1, 1:30 (Guo and Kemphues,
1995
); rabbit anti-PAR-2, 1:3
(Boyd et al., 1996
); mouse
anti-PAR-3, 1:20 (this study); rabbit anti-PAR-6, 1:20
(Hung and Kemphues, 1999
);
mouse anti-PIE-1, 1:10 (Mello et al.,
1996
); rat anti-PKC-3, 1:10
(Tabuse et al., 1998
); rabbit
anti-PGL-1, 1:1000 (Kawasaki et al.,
1998
). Images of immunostained embryos were captured on a
Deltavision microscope (Applied Precision) and deconvolved. Where not
indicated, immunostaining observations were based on the analysis of >15
embryos at the appropriate stage.
Chimeric embryos
Embryos were combined, cultured and immunostained for PAR-3 as described
(Nance and Priess, 2002).
Wild-type and par-3 mutant embryos were combined at the 2-cell stage
such that the anterior cell of the wild-type embryo contacted a par-3
mutant cell. Chimeric embryos were cultured for 2-3 division cycles before
fixation. Wild-type cells were recognized by their distinctive pattern of cell
division and the presence of cortical PAR-3.
Electron microscopy
lon-1 par-3; par-3::zf1::gfp; him-8 and control lon-1
embryos were fixed and processed for electron microscopy as described
(Priess and Hirsh, 1986). For
each genotype, sections of 40-50 fixed embryos at the 12-15 cell stage were
analyzed.
Imaging and analysis of live embryos
Embryos were mounted and imaged for 3D-timelapse microscopy as described
(Nance and Priess, 2002).
Fluorescence images of embryos expressing GFP were acquired on a Leica TCS
scanning confocal microscope.
Reported cell division times are in minutes from the beginning of the
2-cell stage. Times were normalized to those reported by Sulston et al.
(Sulston et al., 1983).
Initiation of ingression of mesodermal cells was scored when these cells first
began to sink below the surface of the embryo.
nmy-1 RNA-mediated interference
Double-stranded RNA (dsRNA) corresponding to bases 5425-5889 of the
nmy-1 cDNA was synthesized as described
(Nance and Priess, 2002).
Young adult hermaphrodites were injected with nmy-1 dsRNA (3.5 µg
µl1) and 24 hours later eggs at the 1-4 cell stage were
collected, mounted and video-recorded for 150 minutes as described above.
Ingression of endodermal cells was analyzed as described in the legend to
Table 1. Endodermal cells
failed to ingress within the recording period in 1 out of 12
nmy-1(RNAi) embryos; reported ingression times are for the remaining
11 embryos. Although it was not possible to monitor NMY-1 protein levels to
quantitatively determine the efficacy of RNAi, all injected control
(lon-1) embryos displayed defects in the elongation stage of
embryogenesis (n=20).
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Results |
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In contrast to the germline precursors, the levels of PAR-3ZF1-GFP and PAR-6ZF1-GFP in somatic precursors diminished to undetectable levels. During the 4-cell stage, when PAR-3, PAR-6 and PAR-6GFP redistributed to the apical cortices of all three somatic precursors (Fig. 1Bc, large arrow, and data not shown), PAR-3ZF1-GFP was absent in the two oldest somatic precursors (the ABa and ABp cells; Fig. 1Bd, large arrow). In EMS, the youngest somatic precursor, PAR-3ZF1-GFP began to redistribute from the cell periphery to the apical cortex (Fig. 1Bd, arrowhead), but disappeared during the following cell cycle. PAR-6ZF1-GFP disappeared similarly in the somatic precursors, but usually required an additional cell cycle to do so (Fig. 1Bf). After the 4-cell or 8-cell stages, PAR-3ZF1-GFP and PAR-6ZF1-GFP could not be detected in the older somatic precursors by either GFP fluorescence or immunostaining for GFP. For example, at the 26-cell stage, when the endodermal precursors normally begin the first cell ingressions, PAR-3ZF1-GFP and PAR-6ZF1-GFP were not detected in the endodermal precursors or the neighboring cells that flank the anterior and lateral sides of the endodermal precursors (Fig. 1Bh and data not shown).
Although normal embryos express PAR-3 and PAR-6 until the 26-cell stage
(Fig. 1Bg and data not shown),
the levels of these proteins gradually decline during subsequent cell cycles.
A second phase of expression coincides with the beginning of organogenesis
(400-cell stage), when PAR-3 and PAR-6 appear in nascent epithelial cells
(Fig. 1Bi and data not shown).
We found that PAR-3ZF1-GFP and PAR-6ZF1-GFP were
expressed in nascent epithelia in a pattern similar to that of endogenous
PAR-3 and PAR-6 (Fig. 1Bj and
data not shown). This result indicates that the machinery that degrades
proteins with the ZF1 domain does not operate at the 400-cell and later
stages. In summary, we conclude that the ZF1 domain effectively removes the
PAR-3ZF1-GFP and PAR-6ZF1-GFP proteins from somatic
precursors between the 4- and 8-cell stages until the beginning of
organogenesis.
par::zf1::gfp transgenes restore anterior-posterior
asymmetry to par mutant embryos
To test whether the PAR-3ZF1-GFP and PAR-6ZF1-GFP
proteins could provide PAR functions essential for anterior-posterior
asymmetry, we crossed the corresponding transgenes into par-3 or
par-6 mutant strains, which lack detectable maternal PAR-3 or PAR-6,
respectively (Etemad-Moghadam et al.,
1995; Hung and Kemphues,
1999
). For simplicity, we refer to a par-3 mutant with an
integrated par-3::zf1::gfp transgene as par-3(ZF1), and to
par-3(ZF1) and par-6(ZF1) embryos collectively as
par(ZF1) embryos. Although the par-3(ZF1) and
par-6(ZF1) embryos had abnormalities in the appearance of early
embryonic cells and in cell movements during gastrulation (see below), most of
the embryos developed to hatching and grew to fertile adults [eggs hatched in
wild type, 1271/1281 (99%); par-3(ZF1), 1061/1085 (98%);
par-6(ZF1), 2443/2548 (96%)]. The par-6(ZF1) strain with the
lowest level of PAR-6ZF1-GFP expression (zuIs52, see
Table 1) produced some embryos
that grew to agametic adults (30/396). This phenotype has been described in
strains with hypomorphic alleles of par-3 and par-6, and is
likely to reflect suboptimal levels of PAR-6ZF1-GFP in germline
cells (Kemphues et al., 1988
;
Watts et al., 1996
).
PAR-6ZF1-GFP was expressed asymmetrically at the 1-cell stage and in the germline precursors in par-6(ZF1) embryos and wild-type embryos, and similar results were observed for PAR-3ZF1-GFP in par-3(ZF1) embryos (Fig. 1Ba,b and data not shown). Thus, the ZF1-tagged proteins provide sufficient par-6(+) or par-3(+) function to promote their own asymmetric localization. We asked whether the anterior-posterior asymmetries that characterize the first division of a wild-type embryo occurred in the par(ZF1) embryos. The first division is unequal in wild-type embryos (Fig. 2A), resulting in a small posterior daughter (the germline precursor), but in par-3 and par-6 mutant embryos the first division is equal (Fig. 2E, Table 1). In all par(ZF1) embryos examined the first cell division was unequal, as in wild-type embryos (Fig. 2I, Table 1). Similarly, the subsequent three divisions of the germline precursors were unequal in the par(ZF1) embryos, as in wild-type embryos (wild type, n=14; par-3(ZF1), n=15; par-6(ZF1), n=14). In wild-type embryos, the first division results in the asymmetric localization of PIE-1 and cytoplasmic granules, called P granules, to the posterior daughter (Fig. 2B,C). These asymmetries are absent in par-3 and par-6 mutant embryos (Fig. 2F,G), but were present in all par-3(ZF1) and par-6(ZF1) embryos (Fig. 2J,K) (P granules: wild type, n=54; par-3(ZF1), n=26; par-6(ZF1), n=62. PIE-1: wild type, n=58; par-3(ZF1), n=35; par-6(ZF1), n=44).
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PAR localization in par(ZF1) embryos
PARZF1-GFP proteins were degraded rapidly in the somatic
precursor cells of the par(ZF1) strains in a pattern similar to the
degradation of PARZF1-GFP proteins in otherwise wild-type strains
(Fig. 1Bc-h, Fig. 3C). Because degradation
of the PARZF1-GFP proteins occurred progressively with the age of
the somatic precursor, we observed several examples where a cell with apical
PARZF1-GFP was adjacent to a cell that lacked
PARZF1-GFP. This pattern indicates that apical restriction of PAR-3
does not require PAR-3-mediated interactions with neighboring cells. To test
this hypothesis further, we combined wild-type embryos with par-3
mutant embryos at the 2-cell stage and allowed the chimeric embryos to divide
in culture. In each of seven chimeric wild-type/par-3 mutant embryos,
PAR-3 was excluded from all surfaces where a wild-type cell contacted a
par-3 mutant cell. Instead, PAR-3 was concentrated on the
contact-free surfaces of the wild-type cells
(Fig. 1Bk,l and Discussion).
These results are similar to those of a previous study where wild-type embryos
were combined with wild-type embryos
(Nance and Priess, 2002) (data
not shown).
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We next asked if the apical localizations of PAR-3, PAR-6 and PKC-3 were interdependent. The youngest somatic cells of par-3(ZF1) embryos contained apical PAR-3ZF1-GFP (Fig. 4C, arrow) and PKC-3 colocalized with PAR-3ZF1-GFP (Fig. 4D, arrow). The older somatic cells lacked PAR-3ZF1-GFP and contained cytoplasmic rather than apical PKC-3 (Fig. 4D). Similarly, PKC-3 also failed to localize to the apical cortex in the somatic cells lacking PAR-6ZF1-GFP in par-6(ZF1) embryos (Fig. 4E,F). By contrast, endogenous PAR-3 showed a robust association with the apical cortex of cells lacking PAR-6ZF1-GFP in par-6(ZF1) embryos (Fig. 4G,H). The apical localization of PAR-3 in the par-6(ZF1) strain does not result simply from the perdurance of cortical PAR-3 after the degradation of PAR-6ZF1-GFP: in both wild-type embryos and in par-6(ZF1) embryos, PAR-3 disappeared transiently from the cortex during cell division and reappeared during the next cell cycle. In summary, PAR-3, but not PKC-3, associates specifically with the apical cortex in cells that lack PAR-6. However, neither PAR-6 nor PKC-3 can associate with the apical cortex of cells that lack PAR-3.
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The first cells to ingress during gastrulation are the two endodermal
precursors. The endodermal precursors move from the ventral surface into the
interior, beginning 90 minutes after the first cleavage of the embryo
(Sulston et al., 1983
). During
ingression, neighboring cells spread across the apical surfaces of the
endodermal precursors (Lee and Goldstein,
2003
; Nance and Priess,
2002
). We found that the ingression of the endodermal precursors
in par(ZF1) embryos was markedly slower than wild-type endodermal
precursors. For example, at a time when wild-type endodermal precursors had
moved 2±1.2 µm (n=5) away from the ventral surface, the
endodermal precursors in par-3(ZF1) embryos had either not moved or
moved only 0.4±0.5 µm (n=6, P<0.05). The
endodermal precursors in wild-type embryos completed ingression in 23 minutes,
which corresponds to a single cell cycle
(Table 1,
Fig. 6Aa-c, Movie 1A at
http://dev.biologists.org/supplemental/).
By contrast, the endodermal precursors in par-3(ZF1) and
par-6(ZF1) strains required 51 and 43 minutes to complete ingression,
respectively, and invariably divided before ingression was complete
(Table 1, Fig. 6Ad-f, Movie 1B at
http://dev.biologists.org/supplemental/).
Expression of PAR-3ZF1-GFP or PAR-6ZF1-GFP in otherwise
wild-type embryos did not slow ingression, indicating that the ingression
defect was not caused by overexpression of the PAR proteins from the
transgenes (Table 1).
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Two genes in C. elegans, nmy-2 and nmy-1, encode
nonmuscle myosin heavy chains. Although nmy-2 is required for
anterior-posterior polarity and cell division, roles for nmy-1 in the
early embryo have not been described (Guo
and Kemphues, 1996). Because endodermal cells in
par-3(ZF1) embryos eventually ingress, we wondered if NMY-1 might
compensate for the reduced levels of apical NMY-2. However, the pattern of
endodermal cell ingression was not altered by depleting NMY-1 by RNA-mediated
interference and endodermal cells were internalized at 136±6.7 minutes
after the 2-cell stage (n=11, compare to par-3(ZF1) and wild
type in Table 1).
In normal development, ingression of the endodermal precursors is followed
by ingression of a group of mesodermal cells that are descendants of an early
embryonic cell called MS. Ingression of these MS descendants begins 1
hour after the E daughters begin their ingression
(Nance and Priess, 2002
;
Sulston et al., 1983
). We
compared the ingression of a pair of MS descendants (MSaaaa and MSaaap) in
wild-type embryos and par-3(ZF1) embryos. In wild-type embryos, both
cells ingressed
11 minutes after their birth (11±2 minutes,
n=5). In each of six par-3(ZF1) embryos examined, the same
cells had variable defects in ingression. In one embryo, neither cell
ingressed during an observation period of two cell cycles. In embryos where
the cells eventually ingressed, the first ingression movements were evident 7
minutes later than in wild-type embryos (18±3 minutes after their
birth, n=5, P<0.05). Thus ingressions in both endodermal
and mesodermal lineages occur more slowly in par-3(ZF1) embryos than
in wild-type embryos.
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Discussion |
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De novo establishment of apicobasal asymmetry within early embryonic
cells
During the 4-cell stage of embryogenesis, the PAR proteins undergo a
dramatic redistribution along the apicobasal axis. Our results indicate that
recruitment of PAR-3 to the apical cortex is a key step in this
redistribution, analogous to previous observations on the role of PAR-3 at the
1-cell stage (Watts et al.,
1996; Tabuse et al.,
1998
). We showed that PAR-3 localization to the apical cortex
occurs independently of PAR-6 (this study) and PAR-2
(Nance and Priess, 2002
).
Moreover, PAR-3 localization is crucial for recruiting PAR-6 and PKC-3 to the
apical cortex, and restricting PAR-2 to basolateral surfaces. Localization of
PAR-3 to the apical cortex is not sufficient for the colocalization of PAR-6
and PKC-3: PAR-6 does not colocalize with apical PAR-3 in pkc-3(RNAi)
embryos (J.N., E.M.M. and J.R.P., unpublished), and PKC-3 does not colocalize
with apical PAR-3 in par-6(ZF1) embryos. Thus both PAR-6 and PKC-3
must be present for either protein to associate with apical PAR-3. Biochemical
studies of PAR-3, PAR-6 and PKC-3 homologs in mammalian cells have shown that
these proteins can bind to one another directly
(Joberty et al., 2000
;
Lin et al., 2000
), indicating
that interactions between all three proteins might be necessary to stabilize a
complex with apical PAR-3.
How is PAR-3 recruited to the apical cortex? Our previous experiments with
recombined embryonic cells demonstrated that PAR-3 is excluded from surfaces
that are in contact with neighboring cells
(Nance and Priess, 2002).
Thus, in a normal embryo PAR-3 would be restricted to the contact-free, apical
surface. We have shown here that the exclusion of PAR-3 from contact surfaces
is not dependent on the presence of PAR-3 in the neighboring cells. First,
par-3(ZF1) embryos can contain young somatic cells with apically
restricted PAR-3ZF1-GFP that are adjacent to older somatic cells
that lack PAR-3ZF1-GFP. Second, wild-type cells that are recombined
with par-3 mutant cells correctly localize PAR-3 to their
contact-free surfaces. In normal development, PAR-3 must distinguish the
apical surface from contact surfaces at each cell cycle because PAR-3
dissociates from and reassociates with the apical cortex before and after each
cell division, respectively. In principle, non-PAR proteins could define a
location on the apical surface that is maintained during cell division.
However, because there is natural variability in cell contacts during cell
division, and because our cell recombination experiments show that the
apicobasal axis is not fixed (Nance and
Priess, 2002
), we favor the hypothesis that the apical surface is
redefined after each of the early cell divisions.
PAR proteins and cell adhesion
The observation that PAR-3 is excluded from contact surfaces indicates that
proteins that are involved in cell adhesion either directly or indirectly
influence PAR-3 localization. In a reciprocal manner, the apical PAR-3 complex
appears to either directly or indirectly modulate cell adhesion. We have shown
that par(ZF1) embryos can develop prominent gaps between the lateral
surfaces of cells, whereas these surfaces are tightly adherent in normal
embryos. Similarly, embryo culture experiments have shown that apical surfaces
have the potential to adhere on contact with other cells
(Nance and Priess, 2002).
Thus, the apical PAR-3 complex might concentrate or modify adhesive factors at
the apical and adjacent lateral surfaces. Alternatively, the apical PAR
complex might direct the vectorial transport to the basal surface of proteins
that inhibit cell adhesion; such proteins would mislocalize to lateral
surfaces in par(ZF1) embryos. By analogy, an apical PAR complex in
Drosophila neuroblasts directs the localization of the Miranda
protein to the opposite end of the cell (reviewed by
Doe and Bowerman, 2001
).
Because cell separations occur between many different types of cells in
par(ZF1) embryos, they appear to be caused by a general defect in
cell-cell adhesion. This defect is unlikely to result from a failure to
transcribe the genes required for cell adhesion because similar separations
are not observed when transcription is inhibited during the early cleavage
stages (Nance and Priess,
2002). Thus, we favor the hypothesis that PAR proteins regulate
either the localization or activity of maternally provided proteins that have
roles in cell adhesion. Maternally expressed HMR-1/E-cadherin associates with
HMP-1/
-catenin and HMP-2/ß-catenin at the basolateral surfaces of
embryonic cells (Costa et al.,
1998
). However, we observed that both HMR-1 and HMP-1 localize
properly in par-3(ZF1) embryos. Depletion of HMR-1 or HMP-1 does not
lead to noticeable defects in the adhesiveness of the early embryonic cells
(Costa et al., 1998
),
indicating that additional adhesive proteins remain to be identified.
Role of the PAR proteins in gastrulation
Defects in cell adhesion could contribute to the abnormally slow cell
ingressions observed in par(ZF1) embryos. When an ingressing cell
separates from its neighbors at the surface of a normal embryo, a transient
gap is created that is closed by the rapid spreading of neighboring cells.
This spreading presumably is mediated by lateral adhesion between the newly
exposed surfaces of the neighboring cells. Thus, adhesion between the
neighboring cells might exert a squeezing force on the ingressing cell that
contributes to the normal speed of ingression.
Defects in apical contraction are a second likely cause of the slowed cell
ingressions of par(ZF1) embryos. In normal embryos, NMY-2/nonmuscle
myosin concentrates at the apical cortex of ingressing cells, reaching a level
comparable to that found in the cleavage furrows of dividing cells
(Nance and Priess, 2002 and
this study). The apical contraction of an ingressing cell could concentrate a
fixed, but initially dispersed, population of cortical NMY-2, and lead to an
apparent increase in the level of NMY-2. However, membrane-associated proteins
such as LAD-1 do not show a similar behavior during cell ingression, raising
the possibility that additional NMY-2 is recruited to the apical surfaces of
ingressing cells (Chen et al.,
2001
) (J.N., E.M.M. and J.R.P., unpublished). Irrespective of the
mechanism by which NMY-2 is concentrated in normal ingression, this
concentration is either markedly reduced or does not occur in
par(ZF1) embryos. Because myosin activity is essential for apical
contraction and ingression (Lee and
Goldstein, 2003
), failure to either concentrate or activate NMY-2
is likely to lead to defects in ingression.
Why doesn't cell ingression fail completely in the par(ZF1)
embryos? It is possible that a small but significant amount of
PARZF1-GFP persists in ingressing cells. Although we cannot
eliminate this possibility, the residual level would have to be below the
level of detection of immunocytochemistry using antibodies against either GFP
or the various PAR proteins. Moreover, all of the lines of transgenic animals
we generated had identical defects in ingression despite considerable
variation in their initial level of PARZF1-GFP at the 1-cell stage.
A second possibility is that the low level of NMY-2 remaining at the apical
surface is sufficient for a weak contraction and slow ingression. It is
unlikely that NMY-1 functions redundantly with NMY-2 in this process because
depleting NMY-1 in par-3(ZF1) embryos does not further impair
endodermal cell ingression. Last, it is possible that the PAR-dependent
concentration of NMY-2 at the apical surface functions primarily to increase
the efficiency of an otherwise PAR-independent pathway for ingression. Cells
that lack PARZF1-GFP proteins retain at least one apicobasal
asymmetry, the basolateral localization of HMR-1/E-cadherin, and basal
localization of structures such as lamellipodia or filopodia could contribute
to ingression. Studies in other systems have shown that the ARP2/3 complex of
proteins functions in the nucleation and branching of microfilaments and that
it localizes to the leading edges of crawling cells (reviewed by
Higgs and Pollard, 2001).
Interestingly, depletion of the C. elegans ARP2/3 complex prevents
ingression of the endodermal precursors
(Severson et al., 2002
). Large
filopodial-like projections are apparent on the C. elegans endodermal
precursors after ingression (J.N., E.M.M. and J.R.P., unpublished), however it
is not known whether smaller filopodia and lamellipodia are present during
ingression. The possibility that multiple mechanisms contribute to cell
ingression in C. elegans is reminiscent of studies on invagination in
Drosophila. During Drosophila gastrulation, invaginating
sheets of cells flatten and contract their apical surfaces, and apical
contraction is associated with an apical accumulation of nonmuscle myosin
(reviewed by Leptin, 1999
).
These shape changes are regulated in part by the Folded gastrulation (Fog)
signaling pathway. However, mutations that disrupt the Fog pathway slow, but
do not prevent, invagination (Costa et al.,
1994
; Parks and Wieschaus,
1991
).
The fact that cell ingressions can be slowed in par(ZF1) embryos
in C. elegans without causing embryonic lethality is surprising given
the essentially invariant positions of embryonic cells during normal tissue
morphogenesis (Sulston et al.,
1983). However, examples of natural variability in cell positions
have been documented in wild-type embryos, where the mispositioned cells can
migrate to their normal location (Schnabel
et al., 1997
). In addition, mutations that block cell death result
in embryos with mispositioned cells. These embryos develop into viable animals
that appear superficially normal, although they have numerous defects in
cellular anatomy (White et al.,
1991
). We do not yet know whether the mispositioned cells in
par(ZF1) embryos undergo compensatory migrations or whether the
resulting animals have anatomical defects that are not apparent by light
microscopy.
Cues and roles for PAR asymmetry
C. elegans embryos have at least three distinct periods in which
the PAR-3 complex must distinguish different cell surfaces. At the 1-cell
stage PAR-3 associates with the anterior surface, and at the 4-cell stage
PAR-3 associates with the apical surface. In late embryogenesis PAR-3 is
localized asymmetrically in epithelial cells, and the apicobasal axis of the
internal epithelia is inverted with respect to that of earlier embryonic cells
(Leung et al., 1999;
McMahon et al., 2001
) (J.N.,
E.M.M. and J.R.P., unpublished). These localization patterns appear to be
specified de novo during each period. Disruption of PAR asymmetry at the
1-cell stage by mutations in par-2 does not prevent apical
localization of PAR-3 after the 4-cell stage
(Nance and Priess, 2002
).
Similarly, we showed that the absence of the PAR-3 complex between the
4-400-cell stages in par-3(ZF1) embryos does not prevent the
subsequent apical localization of PAR-3 during organogenesis.
The molecular cues used to localize the PAR-3 complex remain to be
identified and, at some level, these are likely to vary. For example, sperm
position and cell contacts specify polarity at the 1- and 4-cell stages,
respectively. Although the mechanism of PAR localization has not been studied
extensively in the epithelial cells of C. elegans, genetic studies in
Drosophila have identified homologs of proteins in the C.
elegans PAR-3 complex that regulate apicobasal polarity in epithelial
cells. E-cadherin-mediated cell adhesion is required for apical PAR-3 complex
localization in Drosophila epithelial cells
(Bilder et al., 2003), whereas
HMR-1/E-cadherin is not essential for PAR-3 complex asymmetry at either the
1-cell or 4-cell stage in C. elegans
(Costa et al., 1997
;
Nance and Priess, 2002
).
Apical localization of the PAR-3 complex in Drosophila epithelia is
antagonized by a basolateral complex of proteins that includes Discs large and
Scribble (Bilder et al., 2003
;
Tanentzapf and Tepass, 2003
).
The C. elegans homologs of the latter proteins, DLG-1/Discs large and
LET-413/Scribble, are expressed in epithelial cells, and depletion of these
proteins causes epithelial defects (reviewed by
Knust and Bossinger, 2003
).
However, these proteins do not appear to function in apicobasal polarity of
early embryonic cells because they are either not expressed in the early
embryo (DLG-1) or are not required for apical localization of PAR-3
(let-413) (J.N., E.M.M. and J.R.P., unpublished). Thus, identifying
the molecular basis of cell-contact-dependent PAR localization remains an
important goal for future studies on apicobasal PAR asymmetry.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Howard Hughes Medical Institute, Seattle, WA 98109, USA
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