1 Department of Molecular Biology and Genetics, Cornell University, 107
Biotechnology Building, Ithaca, NY 14853, USA
2 CNRS-CGM, Avenue de la Terasse, 91198 Gif-sur-Yvette, France
Author for correspondence (e-mail:
kjk1{at}cornell.edu)
Accepted 26 February 2004
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SUMMARY |
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Key words: Cell polarity, Ovulation, Gonadogenesis, Caenorhabditis elegans
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Introduction |
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One of these complexes, the PAR-3/PAR-6/aPKC complex, plays a fundamental
role in establishing cell polarities essential for asymmetric divisions in the
early embryo in C. elegans (for reviews, see
Kemphues and Strome, 1997;
Pellettieri and Seydoux,
2002
), but to date there has been no evidence for a role in
epithelial polarity in this animal. Based on the conservation of the
biochemical interactions between PAR-3 and PAR-6 family members and their
conserved role in epithelial polarity in flies and mammals
(Izumi et al., 1998
;
Johnson and Wodarz, 2003
;
Knust and Bossinger, 2002
;
Kuchinke et al., 1998
;
Ohno, 2001
), it seems
reasonable to expect a role for the complex in epithelial polarity in C.
elegans as well. The proteins are expressed and co-localized apically in
C. elegans gut and vulva (Hurd
and Kemphues, 2003
; Leung et
al., 1999
; McMahon et al.,
2001
), consistent with a role in one or both of these polarized
epithelia. However, none of the existing mutants in par-3 and
par-6, including amber mutations in par-3
(Cheng et al., 1995
;
Watts et al., 1996
), results
in obvious defects in polarized epithelia. It is possible that roles in
epithelial development are masked by persistence of wild-type maternal mRNA
and protein in homozygous progeny derived from heterozygous mothers and by the
polarity defect in early embryos produced by homozygous mutant mothers.
Indeed, it has recently been shown that PAR-3 has a role in cell adhesion and
gastrulation in the early embryo that is only revealed when the early polarity
requirement is bypassed (Nance et al.,
2003
). To explore a possible role for PAR-3 in vulval development,
we depleted PAR-3 post-embryonically using RNAi.
Unexpectedly, we found that PAR-3-depleted worms exhibit defects in ovulation and fail to store sperm properly due to defective functioning of the spermatheca. The spermatheca is a tubular epithelium that acts in sperm storage, ovulation and fertilization. We discovered that PAR-3 is transiently expressed and localized asymmetrically at or near apical junctions in spermathecal precursor cells of L4 larvae. We also found that the cell polarity of a subset of cells in the distal spermatheca is severely affected in PAR-3 depleted worms. Finally, we determined that the ovulation defects of par-3(RNAi) worms can be suppressed by a mutation that can bypass defects in signaling between the oocyte and the spermatheca. From these observations, we propose that PAR-3 activity is necessary for the proper polarization of cells in the distal spermatheca and that defective ovulation and storage of mature sperm result from failure of the distal spermathecal cells to respond to signals from the oocyte that trigger spermathecal dilation.
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Materials and methods |
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RNAi
In initial experiments, we tested independently the NH2-terminal
1.6 kb fragment of par-3 (GenBank Accession Number U25032)
(nucleotides 1-1686 (X1)) and three internal fragments obtained by restriction
digest of HindIII (nucleotides 840-1888 (H1), 1889-2755 (H2) and
2756-4277 (H3)). Throughout this study, we used the NH2-terminal
construct as it caused the most consistent, highly penetrant Emo phenotype.
For RNAi of par-6 (GenBank Accession Number AF070968) and
pkc-3 (GenBank Accession Number AF025666), we used full-length cDNA
clones. Each DNA was inserted into the vector pPD129.36
(Timmons et al., 2001) for
expression of par-3 dsRNA in Escherichia coli strain
HT115(DE3). Induction and feeding were performed as previously described
(Timmons et al., 2001
), except
that 0.5 mM IPTG was used. HT115(DE3) harboring the empty vector served as
control bacteria for the feeding experiments.
Fixation, immunocytochemistry and microscopy
Larvae were fixed in methanol according to standard protocols
(Miller and Shakes, 1995). To
permeabilize prior to fixation, larvae were compressed between two
poly-lysine-coated slides and frozen over dry ice. The slides were rapidly
separated, tearing the cuticle in the process, and then immediately immersed
in 20°C methanol while still frozen. For staining with DAPI,
collected worms were fixed without freezing in 2% paraformaldehyde in
phosphate buffered saline (PBS) for 24 hours at 4°C followed by incubation
with 1 ug/ml DAPI in TBS (150 mM NaCl, 50 mM Tris-HCl, pH 7.5) for 48 hours at
room temperature, and then were mounted on slides for observation. Gonad
dissecting and phalloidin staining were performed as described previously
(McCarter et al., 1997
;
Strome, 1986
). The protocols
in Rose et al. (Rose et al.,
1997
) were applied for the observation of ovulation in vivo and
for staining with anti-myosin antibodies.
Two antibodies to PAR-3 produced identical staining patterns in the somatic
gonad precursor cells of developing larvae. These included a previously
described antiserum raised against the middle portion of PAR-3
(Etemad-Moghadam et al., 1995)
and a monoclonal antibody raised against the same fragment used for the
antiserum (Nance et al.,
2003
). All pictures shown here were obtained with the monoclonal
antibody at a dilution of 1:50. Affinity-purified rabbit polyclonal antibody
to PAR-6 (Hung and Kemphues,
1999
) and affinity-purified rat polyclonal antibody to PKC-3
(Hung and Kemphues, 1999
) were
used at a 1:20 dilution. A new polyclonal antibody to LET-413 was generated
against the central part of the protein (residues 469 to 576) and used at a
dilution of 1:5000. The monoclonal antibody to myosin heavy chain A
(Miller et al., 1986
) and
rabbit polyclonal antibody to CEH-18
(Greenstein et al., 1994
) were
used at 1:50 and 1:200, respectively. Secondary antibodies (Jackson
Immunoresearch) were used at 1:400 (for anti-LET-413) or 1:100 (all
others).
Differential interference contrast epifluorescence microscopy and digital image capture were performed using an Olympus BX60 fitted with a Hamamatsu Orca C4742-95 camera. Confocal laser scanning microscopy was performed on a Leica TCSSP2. Final figures were assembled using Photoshop (Adobe Systems).
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Results |
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A comparison of par-3(RNAi) and control worms revealed a slightly
reduced growth rate in the treated individuals. On average, the treated worms
molted into adults 2-3 hours later than controls. This difference is within
the range of the developmental variation within each culture. The RNAi-treated
worms were as motile as wild-type worms, suggesting that the neuromuscular
system developed normally. However, the reproductive system seemed to be
damaged severely, as few eggs were laid by par-3(RNAi) worms. As this
phenotype had not been reported previously after analysis of par-3
mutant worms (Cheng et al.,
1995), we decided to examine this further. First, we compared the
number of embryos present on plates containing 10 control versus 10 RNAi worms
after 48 hours of growth. The control plate had >500 embryos, whereas the
par-3(RNAi) plate had only 70. These results suggest that PAR-3
depletion affects oogenesis or ovulation. Supporting this hypothesis, the most
proximal part of the ovary was distended (data not shown). To investigate the
phenotype further, we visualized the DNA in oocytes by staining with DAPI and
noted that these worms showed an endomitotic oocyte (Emo) phenotype
(n>50) (Fig. 1B)
(Iwasaki et al., 1996
). This
phenotype commonly arises through defects in ovulation.
|
In addition to the Emo phenotype, we noted that par-3(RNAi) worms
failed to accumulate sperm in the spermatheca; instead sperm accumulated at
the extreme proximal portion of the ovary (n>50 worms)
(Fig. 1B). In normal
hermaphrodites, sperm are born at the proximal end of the ovotestis during L4
and become localized to the spermatheca by migration or are swept in during
the first ovulation (Ward and Carrel,
1979).
These phenotypes are the result of PAR-3 depletion. RNAi using different non-overlapping fragments of par-3 cDNA gave similar results (43/47 with fragment X1, 37/97 with H1, 31/93 with H2 and 77/103 with H3 showed an Emo phenotype; see Materials and methods). Furthermore, as we describe in detail below, PAR-3 protein was expressed in gonadal cells during their development and was depleted by RNAi.
par-3(RNAi) worms show defects in ovulation
To determine the basis for the Emo phenotype, we examined oocyte maturation
and ovulation in par-3(RNAi) worms. During normal ovulation, oocyte
maturation, marked by NEBD, is followed by intensive contraction of sheath
cells. Oocytes enter the spermatheca within 2 minutes of NEBD (n=5)
(Fig. 2, left column). In
RNAi-treated worms, NEBD and contraction of sheath cells occurred as in
controls, but oocytes had not entered the spermatheca even 4 minutes after
NEBD (n=4) (Fig. 2,
right column). Failure to ovulate in spite of vigorous sheath contractions,
combined with the observation of mislocalized sperm in adult worms, suggests
that the spermatheca, rather than gonad sheath, is not functional in
par-3(RNAi) worms.
|
Gonad sheath cells develop normally in par-3(RNAi) worms
Vigorous sheath cell contractions suggested that the gonad sheath cells
were functioning normally after par-3 RNAi. As a second assay for
sheath cell development, we analyzed the expression of sheath-cell-specific
markers. Each gonad sheath consists of five pairs of cells arranged in a
reproducible pattern and expressing the transcription factor ceh-18
in the nucleus and myosin heavy chain A (MHCA) in an organized cytoplasmic
array (Greenstein et al., 1994;
Rose et al., 1997
). We
compared the distribution of CEH-18 and MHCA in wild-type and
par-3(RNAi) gonads dissected from worms grown for 48 hours from L1
(young adults). We detected no difference from wild type in the number of
sheath cells or in the expression or distribution of the sheath cell markers
(for CEH-18, n=12 control and n=16 par-3(RNAi)
worms) (Fig. 3A,B) (for MHCA,
n=50 par-3(RNAi) worms)
(Fig. 3C,D).
|
Spermathecal cell numbers and cell fates are not affected in par-3(RNAi) worms
During early embryogenesis, PAR-3 contributes to asymmetric cell division
and cell fate specification. Therefore, we considered the possibility that
par-3 RNAi affected asymmetric division and cell fate specification
in the cell lineage leading to the spermatheca. To address the question of
whether asymmetric cell divisions were normal, we counted the number of cells
in the entire spermatheca after staining nuclear DNA by 0.01 mg/ml of
propidium iodide. If the cell division pattern was disorganized, the number of
cells in developed tissue could be different from the wild type. However, all
nine spermatheca examined contained the expected 30 cells
(Kimble and Hirsh, 1979).
To test for proper cell fate specification, we observed the expression of
two markers of spermathecal differentiation: GFP fused with the promoters of
let-502, the homolog of rho-associated kinase
(Wissmann et al., 1999) and
ipp-5, inositol 5-phosphatase (Bui
and Sternberg, 2002
). The let-502-driven GFP protein is
expressed in all 30 spermathecal cells, but the level of expression is not
even, with highest levels in the proximal and distal cells
(Wissmann et al., 1999
). In
particular, GFP accumulates to extremely high levels in the four distal-most
cells (Fig. 4). The
ipp-5-driven GFP is expressed in the distal region of the adult
spermatheca (Bui and Sternberg,
2002
). We saw no difference between control and
par-3(RNAi) worms in the expression of these markers (n=21
control and par-3(RNAi) worms for let-502::GFP and
n=27 control and par-3(RNAi) worms for ipp-5::GFP).
These results indicate that the specification of cell type or cell division
pattern seems not to be disturbed by par-3(RNAi).
|
Ovulation in par-3(RNAi) worms is restored by mutation in ipp-5, an inositol polyphosphate 5-phosphatase
Defective signaling between oocyte and spermatheca can lead to failures in
ovulation (Clandinin et al.,
1998). Specifically, the epidermal growth factor (EGF)-like
protein LIN-3, produced in the oocyte, appears to signal through the receptor
tyrosine kinase LET-23 in the spermatheca to trigger spermathecal dilation (J.
McCarter, M-H. Lee and T. Schedl, personal communication). LET-23 appears to
transduce this signal via inositol 1,4,5-triphosphate (IP3)
(Bui and Sternberg, 2002
;
Clandinin et al., 1998
). To
test whether defective signaling might contribute to the Emo phenotype of
par-3(RNAi) worms, we took advantage of the observation that a
mutation in ipp-5, encoding a putative inositol 5-phosphatase, can
suppress mutations in lin-3 and let-23
(Bui and Sternberg, 2002
).
Mutants for ipp-5 have hyperactive spermathecal dilation, presumably
due to aberrant regulation of IP3-mediated calcium release
(Bui and Sternberg, 2002
). For
the experiment, we grew ipp-5(sy605) and ipp-5(+) worms from
L1 larvae on bacteria producing par-3 dsRNA or on control bacteria.
Using a bacterial culture that produced a strong Emo phenotype in the
ipp-5+;par-3(RNAi) worms, we found that
ipp-5;par-3(RNAi) worms were nearly as fecund as
ipp-5 worms fed on control bacteria. During a 2.5-hour egg-laying
interval ipp-5;par-3(RNAi) worms produced an average of 12 eggs per
worm (n=60 worms), whereas ipp-5 worms fed on control
bacteria produced 15 eggs per worm (n=105 worms). As an internal
control for the effectiveness of par-3 RNAi, we scored for maternal
effect lethality. Whereas 80% of the eggs laid by ipp-5 worms fed on
control bacteria hatched, only a single worm hatched from eggs laid by
ipp-5;par-3(RNAi) worms. Thus, ipp-5(sy605) partially
suppresses the Emo phenotype caused by par-3(RNAi). A sample of 29
ipp-5;par-3(RNAi) worms examined under the compound microscope
revealed that 18 were completely suppressed, five were Emo in one gonad arm,
and six were Emo in both arms. Presumably, these latter two classes of worms
account for the reduction in egg production relative to the controls.
The apical organization of distal spermatheca cells is abnormal in par-3(RNAi) worms
In order to clarify the basis for the defects in organization and function
of the distal spermatheca, we examined the distribution of three proteins with
polarized accumulation in epithelial cells: AJM-1, LET-413 and actin.
We first compared the distribution of AJM-1::GFP
(Köppen et al., 2001) in
control and par-3(RNAi) worms grown from L1 at 36, 39 and 42 hours.
The adult spermatheca consists of 30 cells that make up an epithelial tube
with three distinctive regions (McCarter
et al., 1997
). Most proximal to the uterus is the spermathecal
valve, a specialized multinucleate cell through which embryos must pass to
enter the uterus (White,
1988
). The middle region consists of 16 cells with circumferential
actin cables on the basal surface (Strome,
1986
) (Fig. 5E).
AJM-1::GFP appears as an irregular and somewhat compressed meshwork that
outlines the apical junctions between these cells
(Fig. 5A,C). Most distal are a
group of four pairs of cells that form a tube with a high concentration of
microfilaments adjacent to the lumen; the two most distal cell pairs express
high levels of let-502::GFP
(Wissmann et al., 1999
)
(Fig. 4A). These distal cells
must dilate to allow the mature egg to pass into the spermatheca during
ovulation. Just as in the rest of the spermatheca, AJM-1::GFP marks the apical
regions of these cells, forming two parallel lines of signal (arrowheads in
Fig. 5C). We could detect no
discernable and consistent difference between control and par-3(RNAi)
treated worms in AJM-1::GFP signal in the proximal and middle portions of the
spermatheca. However, the distal region of the spermatheca showed consistent
disorganization at all three stages examined, as monitored by AJM-1::GFP
(n>20 in each stage; see Fig.
5B,D). In particular, the two apical lines of AJM-1::GFP were
either missing or disrupted and small patches of AJM-1::GFP signal were
frequently detected far from the apical region of the cells.
|
PAR-3, PAR-6 and PKC-3 are transiently expressed and localized asymmetrically in spermathecal precursor cells
To see the correlation between spermathecal defects and the expression
pattern of PAR-3, we performed immunohistochemistry with antibody to PAR-3.
PAR-3 was detected in the vulval cells, as reported previously
(Hurd and Kemphues, 2003)
(Fig. 6B',C'), as
well as in most or all somatic gonad precursor cells, including ancestors of
uterus, sheath, and spermatheca starting during the third larval stage (L3;
about 25-29 hours at 25°C). Figure
6 shows a time-course of the accumulation of PAR-3 and AJM-1
protein during the development of the somatic gonad. PAR-3 began to accumulate
in developing uterine, sheath and spermathecal cells during the late-L3 stage.
At this stage, PAR-3 appeared to accumulate below the cell membranes that face
neighboring cells (Fig. 6A). At
this time very little AJM-1 was detected, and what was present was not yet
concentrated at apical junctions (compare gonadal cells with vulval cells in
Fig. 6A). As development
proceeded, asymmetric distribution of PAR-3 became evident
(Fig. 6B,C). By the mid-L4
stage most somatic gonad precursors expressed PAR-3, with the exception of
cells at the boundary of the uterus and the spermatheca, which we presumed to
be precursors of the spemathecal valve
(Fig. 6C). Only in mid-L4 did
AJM-1 begin to accumulate apically, starting in the proximal gonad and moving
more distally (Fig.
6C'',D''). Interestingly, the appearance of AJM-1 at
apical junctions roughly correlated with the disappearance of PAR-3
(Fig. 6D).
|
|
To determine if this relationship holds true at the functional level, we extended our analysis to examine the effect of par-6 RNAi and pkc-3 RNAi on ovulation. For both RNAi experiments, worms grown for 48 hours on RNAi feeding bacteria were stained with DAPI and scored for the Emo phenotype. For par-6 RNAi, 41/50 worms showed an Emo phenotype; for pkc-3 RNAi, 17/48 worms showed an Emo phenotype. In the same experiment, 43/47 par-3(RNAi) worms and 0/20 control worms were Emo. These findings suggest that PAR-3/PAR-6/PKC-3 act together in the distal spermatheca.
PAR-3 is expressed in somatic gonad precursor cells of par-3 mutant animals
All existing par-3 mutations are maternal-effect-lethal mutations;
par-3/ progeny of par-3+/ mothers grow
up to become adults that produce inviable embryos. These worms do not exhibit
Emo phenotypes. One of these mutations, it71, by genetic and
immunological criteria, was judged to be a probable null allele
(Cheng et al., 1995;
Etemad-Moghadam et al., 1995
).
Consistent with previous genetic results, we found that the it71
sequence contained an amber mutation at amino acid position 83. We sequenced a
second par-3 allele, t1591, and found that it also contained
a nonsense mutation in the extreme amino terminus of the protein (position
38). To address the reason that these alleles do not exhibit an Emo phenotype,
we examined PAR-3 accumulation in par-3(it71) homozygous larvae. We
found that although embryos from par-3(it71) mothers fail to
accumulate PAR-3 protein (Etemad-Moghadam
et al., 1995
), the mothers themselves, as larvae, accumulate PAR-3
at normal levels in the developing somatic gonad (n>20)
(Fig. 7G). Thus it is possible
that all existing par-3 mutations reside in regions of the protein
that are only expressed or required maternally.
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Discussion |
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PAR-3 is required for spermathecal function
Worms treated with par-3 RNAi during larval life are sterile and
exhibit an Emo phenotype, in which oocytes accumulate in the proximal ovary
with endomitotic nuclei. We determined, using rrf-1, a mutant in
which RNAi is blocked in the soma but not the germline
(Sijen et al., 2001), that the
Emo phenotype resulted mainly, perhaps entirely, from a defect in the soma,
most likely a defect in ovulation. Direct observations of defective first
ovulations in par-3(RNAi) worms confirmed this.
Successful ovulation requires contractions of the sheath cells in the
proximal gonad in conjunction with dilation of the distal spermatheca
(McCarter et al., 1997;
Rose et al., 1997
). Three
observations suggest that the basis for defective ovulation after
par-3 RNAi is a failure in spermathecal function. First, sheath cells
undergo extensive contraction, are organized normally and express appropriate
differentiation markers. Second, in spite of extensive sheath cell
contractions, the spermatheca fails to dilate to cover the mature eggs. Third,
sperm accumulate exclusively in the proximal gonad and are not detected in the
spermathecae of affected worms.
Our microscopic examination of spermathecae revealed clear morphological abnormalities in the four distal-most cells. These cells pair to form a narrow tube in control animals. However, in par-3(RNAi) worms, the distal cells are more randomly positioned with respect to each other and fail to form the tight arrangement observed in the wild type. This raised the possibility that the spermatheca was physically blocked, preventing passage of eggs. However, as described below, we found that this was not the case in most Emo worms.
PAR-3 depletion results in defective oocyte/spermatheca signaling
In normal worms, dilation of the spermatheca requires signaling from the
oocyte to the spermatheca that depends upon the epidermal-growth-factor type
ligand LIN-3 and the receptor tyrosine kinase LET-23 and upon components of
the inositol phosphate signaling system
(Bui and Sternberg, 2002;
Clandinin et al., 1998
).
Reduction-of-function mutations in let-23 and lin-3 that
cause Emo phenotypes can be suppressed by loss-of-function mutations in
ipp-5, a gene encoding a type I 5-phosphatase. Like other
5-phosphatases (Majerus,
1992
), IPP-5 presumably negatively regulates inositol signaling by
dephosphorylating 1,4,5 inositol triphosphate
(Bui and Sternberg, 2002
). We
found that the ipp-5 mutation effectively suppressed the effects of
PAR-3 depletion, arguing strongly that the Emo phenotype results from a defect
in signaling between the oocyte and the spermatheca. The incomplete
suppression of the Emo phenotype in par-3(RNAi) worms, in contrast to
the strong suppression of let-23 and lin-3 mutants
(Bui and Sternberg, 2002
),
however, raises the possibility that some par-3(RNAi) worms might
have structural defects that cannot be suppressed by ipp-5.
Apical organization of the distal spermathecal cells require PAR-3
We propose that the primary defect caused by par-3 RNAi is in the
organization of the apical domain of the distal spermathecal cells. Although
we cannot rule out a subtle defect in cell fate specification or in the
asymmetric divisions leading to the formation of the spermatheca,
par-3 RNAi has no detectable effect on cell numbers or on the
expression of cell fate markers. We observed that PAR-3 starts to accumulate
in the late-L3 stage and localizes to the apical regions prior to AJM-1, a
marker for apical organization. This suggests that PAR-3 could play a role
early in the polarization of this tissue. In fact, this idea is supported by
our finding that removal of PAR-3 from precursors in the distal half of the
spermatheca resulted in mislocalization of AJM-1 and apical microfilaments in
these cells. Suppression by ipp-5 argues that a consequence of apical
disorganization is a mislocalization of the signal transduction machinery in
the distal spermathecal cells.
The effect of PAR-3 depletion on oocyte/spermathecal signaling is
strikingly reminiscent of the effect of mutations in lin-2, lin-7 and
lin-10 on LIN-3/LET-23 signaling during vulval development
(Kaech et al., 1998). In the
vulva, LIN-2, LIN-7 and LIN-10 proteins act in a complex to ensure the
basolateral accumulation of LET-23; inactivation of the complex results in
failure of vulval induction (Kaech et al.,
1998
). Unfortunately, we were unable to detect LET-23 protein in
the distal spermatheca to assess whether LET-23 distribution was affected by
depletion of PAR-3.
A restricted role for PAR-3 in the distal spermatheca?
It is puzzling that in spite of widespread expression of PAR-3 in the
somatic gonad, defects are only observable at the extreme distal end of the
spermatheca. Although this could reflect the only requirement for PAR-3 in
post-embryonic development, it is also possible that par-3 RNAi is
ineffective in revealing a more general post-embryonic requirement for PAR-3.
We propose that the more restricted par-3(RNAi) phenotype arises as a
combination of incomplete depletion of the protein at early stages of gonadal
development, the relatively late development of the distal spermatheca and the
absence of a role for PAR-3 in maintenance of the apical organization. Gonadal
development proceeds from proximal to distal tissues, with proximal cells
undergoing terminal differentiation while the distal cells are still
proliferating (Kimble and Hirsh,
1979). In particular, we note that
-catenin and AJM-1 are
recruited into apical junctions in a proximal-distal direction (see
Fig. 6C'',D'' and
Fig. 7A''). Inversely
correlating with this difference, after RNAi, PAR-3 is first depleted from the
distal spermathecal cells, perhaps because the newly formed apical junctions
in the proximal cells block the rapid turnover of the protein. By the time
that the bulk of the PAR-3 protein has been depleted from the more proximal
gonad, PAR-3 might no longer be required. This possibility is suggested by
observations that dominant negative mutations in aPKC and PAR-6 affect
establishment but not maintenance of polarity in MDCK cells
(Suzuki et al., 2001
;
Yamanaka et al., 2001
).
An unexpected result of our analysis is the existence of PAR-3 proteins in
homozygous par-3(it71) larvae. This allele has a nonsense mutation at
the 83rd codon. Our antibody was raised against the central domain of PAR-3,
and no protein is detected in embryos produced by homozygous worms
(Etemad-Moghadam et al., 1995).
There are two possible explanations for the source of the larval signal in the
mutant homozygotes. One possibility is that the maternal supply of wild-type
mRNA is sufficient for both embryonic and post-embryonic development. This
seems unlikely, because the entire somatic gonad (143 cells) arises from only
two blast cells (out of the 558 embryonic cells), and considerable dilution of
the maternal products must take place. The other possibility is that
post-embryonic PAR-3 protein is translated from an mRNA that differs from the
embryonic message and is unaffected by any existing mutation. This is quite
possible, since the screens to identify most existing mutations were
maternal-effect-lethal screens that would not have detected zygotic lethal or
sterile mutations. Unfortunately, no complete par-3 cDNAs are present
in available databases, and our attempts to identify a post-embryonic-specific
mRNA or protein isoform have not yet been successful.
From extensive studies, PAR-3 is known to interact with PAR-6 and PKC-3
during cell polarization in various systems. Here we report observations
providing evidence that PAR-3, PAR-6 and PKC-3 also work together in
organizing apical domains during somatic gonad development in C.
elegans. The three proteins show similar distributions in the developing
somatic gonad, and distributions of PKC-3 and PAR-6 are dependent upon PAR-3,
as shown by par-3 RNAi. Furthermore, removal of PAR-6 and PKC-3 by
RNAi produces a phenotype similar to that of par-3 RNAi. It is
possible that the complex functions in the same way in C. elegans as
it does in other systems. At least one aspect of the relationship between the
PAR-3(Bazooka)/PAR-6/PKC-3 complex and LET-413(Scribble) appears to be
conserved in flies and worms. In the embryonic ectoderm of flies, the
Bazooka/PAR-6/aPKC domain expands in the absence of Scribble, whereas Scribble
distribution is insensitive to removal of Bazooka
(Bilder et al., 2003;
Tanentzapf and Tepass, 2003
).
Similarly, we found that the distribution of LET-413 was normal in distal
spermathecal cells after the removal of PAR-3
(Fig. 5B,D,E,F), and in studies
in the intestinal epithelium the apical domain of the PAR-3/PAR-6/PKC-3
complex was shown to expand after removal of LET-413
(McMahon et al., 2001
).
In summary, PAR-3 accumulates apically in somatic gonad precursor cells. PAR-3 activity is required in the distal spermatheca for the proper localization of AJM-1::GFP, an apical marker, and microfilaments. The loss of polarity in the distal spermatheca correlates with defective ovulation (Emo phenotype) and mislocalization of mature sperm in the adult. We propose that loss of epithelial polarity in the spermathecal cells results in failure to organize the cortical signal transduction system required for spermathecal dilation and might in extreme cases lead to blockage of the spermathecal lumen. Based on these findings, we propose that spermathecal development in C. elegans has the potential to serve as a model system to address the question of how cells acquire asymmetric properties and, in particular, how cells interact to form epithelial tubes.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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