1 Joslin Diabetes Center and Department of Pathology, Harvard Medical School,
One Joslin Place, Boston, MA 02215, USA
2 Laboratory for Germline Development, RIKEN Center for Developmental Biology,
Kobe, Hyogo 650-0047, Japan
* Author for correspondence (e-mail: keith.blackwell{at}joslin.harvard.edu)
Accepted 24 August 2005
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SUMMARY |
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Key words: Germline, Oocyte, Caenorhabditis elegans, Drosophila, RNA binding, Apoptosis, Cytokinesis, P body
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Introduction |
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In metazoa as diverse as C. elegans and humans, one hallmark of
oogenesis is that apoptosis occurs at a high frequency during or shortly after
the late pachytene stage of meiosis (Baker,
1963; Borum, 1961
;
Gumienny et al., 1999
;
Pepling and Spradling, 2001
).
In mammals, developing oocytes transfer cytoplasmic components within cysts,
then, around the time of birth, approximately two-thirds of these oocytes die
as their cysts break down (Pepling and
Spradling, 2001
). About half of all developing oocytes undergo
apoptosis in C. elegans, just before the survivors form discrete
cells from a syncytium (Fig. 1)
(Gumienny et al., 1999
). The
functions of developmental germ cell apoptosis are not well understood. In
C. elegans, this cell death is referred to as physiological because
the sacrificed nuclei do not seem to be of poor quality, and because their
associated cytoplasm is provided to their surviving sisters
(Gumienny et al., 1999
).
Although this suggests that the dying nuclei function in effect as nurse
cells, the absence of physiological apoptosis does not significantly impair
fertility under normal laboratory conditions
(Gumienny et al., 1999
).
It is not known how physiological germ cell death is regulated, but it is
clear that this process is controlled differently from all other apoptosis in
C. elegans, including a pathway that culls defective germ cell nuclei
in response to genotoxic stress (Gartner
et al., 2000; Gumienny et al.,
1999
; Hofmann et al.,
2002
). Whole-genome and other RNAi analyses have identified only
five genes that specifically prevent the physiological apoptosis pathway from
claiming the vast majority of developing oocytes
(Lettre et al., 2004
;
Navarro et al., 2001
), which
suggests that this process is influenced by specific cues.
Another conserved feature of oogenesis is that many newly produced mRNAs
are localized to cytoplasmic storage structures. In many species, the germline
is maintained from one generation to the next by ribonucleoprotein (RNP)
particles that are referred to as germ plasm
(Houston and King, 2000;
Rongo et al., 1997
;
Saffman and Lasko, 1999
;
Wylie, 2000
). For example,
C. elegans germline cells are distinguished by P granules that are
present throughout the life cycle (Strome
and Wood, 1982
). In essentially all metazoa, additional regulatory
complexes that are distinct from germ plasm maintain some oocyte mRNAs in a
deadenylated and translationally quiescent state until these mRNAs are to be
translated (Chang et al., 2004
;
Cao and Richter, 2002
;
Johnstone and Lasko,
2001
).
|
We have now identified a germline RNA-binding protein that we call CAR-1 (cytokinesis/apoptosis/RNA-binding) that associates with CGH-1 within a conserved RNP complex, and in cytoplasmic foci. CAR-1 and CGH-1 orthologs similarly associate in Drosophila oocytes. RNAi knockdown of CAR-1 causes defective embryonic cytokinesis, along with an increase in physiological apoptosis that partially compensates for an oogenesis defect that otherwise leads rapidly to gonad failure. Increased germ cell death plays a similar role after knockdown of the CPEB (cytoplasmic polyadenylation element binding protein) ortholog CBP-3, which interacts functionally with CGH-1 orthologs in other species. We conclude that CAR-1 has a conserved role in germ cell development, and that physiological germline apoptosis may enhance the efficiency of oogenesis, and can partially compensate for a lack of some functions of the CGH-1/CAR-1 complex.
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Materials and methods |
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Antibody production and immunofluorescent staining
Polyclonal antibodies were raised to a CAR-1 peptide (amino acids 246-265,
plus an amino terminal cysteine; NH2-CKAEGKTGRPDWKKERETNQE-COOH) in
two chickens (Cocalico Biologicals, Pennsylvania, USA). Immunostaining with
affinity purified CAR-1 antibody (Sulfolink, Pierce) was reduced to background
in car-1(RNAi) germlines (see Fig. S1 in the supplementary material).
Rabbit anti-CGH-1 antibodies used for affinity purification were generated
against the peptide NH2-CDPKLYVADQQLVDAADETTA-COOH, representing
CGH-1 residues 411-431. Immunostaining was performed using rat anti-CGH-1 and
rabbit anti-PGL-1 (Kawasaki et al.,
1998), as described (Navarro
et al., 2001
). Carnoy's fixative was used to prepare intact worms
for staining with 4',6-diamidino-2-phenylindole (DAPI)
(Villeneuve, 1994
). Nomarski
and fluorescent images were obtained using an Axioskop 2 microscope coupled
with an AxioCam digital camera (Zeiss). Confocal images were obtained using a
Zeiss LSM 510 UV microscope.
Trailerhitch-specific polyclonal antibodies were raised in rabbits against
His6-tagged full-length recombinant protein that was expressed in
E. coli, and purified with Ni-NTA agarose chromatography (Qiagen) and
preparative SDS-PAGE (Kitayama Labes, Nagano, Japan). Drosophila
ovaries expressing EGFP-Me31B (Nakamura et
al., 2001) were immunostained with anti-Trailerhitch antisera and
mouse anti-GFP 3E6 (Wako Pure Chemicals, Osaka, Japan)
(Kobayashi et al., 1999
).
Anti-rabbit IgG Alexa 568 and anti-mouse IgG Alexa 488 (Molecular Probes) were
used as secondary antibodies. Fluorescent images of Drosophila were
acquired using a Leica TCS SP2 AOBS laser confocal microscope.
Co-immunoprecipitation, western analysis and protein identification
C. elegans protein extracts were prepared from 500,000
synchronized hermaphrodites (approximately 12 hours after the L4/adult molt)
by sonication in homogenization buffer [100 mM NaCl, 25 mM HEPES (pH 7.5),
0.25 mM EDTA, 2 mM DTT, 5 mM Na2VO4, 0.1% NP40]
supplemented with 1x`cOmplete' protease inhibitors (Roche) and 50 U/ml
RNasin (Promega), followed by 20 strokes in a glass homogenizer. Homogenates
were centrifuged at 15,000 g for 20 minutes at 4°C, and
the supernatant either used immediately for immunoprecipitation or snap frozen
in liquid nitrogen and stored at 80°C. Protein lysates (1 mg) were
preabsorbed against protein L or G for 1 hour at 4°C. Affinity purified
CAR-1 or CGH-1 antibodies were added to the cleared lysate and incubated for 1
hour at 4°C, after which protein L or G Sepharose beads were added and
incubated for an additional hour. The beads were washed five times in 200 mM
NaCl, 50 mM Tris (pH 7.4), 0.05% NP40, then proteins were extracted by boiling
in 2xSDS sample buffer. To investigate the requirement of RNA for
co-immunoprecipitation, protein lysates were prepared as described except that
5 µg/ml RNase A was added in place of RNAsin, and samples were incubated at
room temperature for 15 minutes before centrifugation. Western blotting was
conducted according to standard procedures, using species-specific
HRP-labelled secondary antibodies (KPL) at a dilution of 1/2500. For mass
spectroscopy, CGH-1 immunoprecipitations were conducted essentially as
described above, but using 5 mg of protein lysate and 25 µg rabbit
anti-CGH-1 or IgG antibodies. Bound proteins were eluted by incubation with
0.2 mg/ml CGH-1 peptide for 1 hour at 4°C. Eluted proteins were resolved
in a 10% polyacrylamide gel that was stained with Simply Blue (Invitrogen).
Excised proteins were digested with trypsin and subjected to tandem mass
spectrometry [Pathology Functional Proteomics Center (PFPC), Harvard Medical
School]. Proteins were identified by searching the NCB Inr database using the
Mascot program. Immunoprecipitation of Drosophila ovary extracts,
western analysis and protein identification were conducted as described
(Nakamura et al., 2004).
|
Analysis of brood size and germ cell death
To measure brood size, L4 stage F1 RNAi hermaphrodites and age-matched N2
and ced-3 animals were individually distributed to NGM plates and
transferred at 12 hour intervals to fresh plates. Progeny were counted
30-40 hours after the removal of the adult.
To measure germ cell death by counting corpses, car-1(RNAi) adults
were grown at 25°C and immobilized in M9 containing 0.03% tetramisole.
Germ cells undergoing apoptosis were then identified by Nomarski optics
(Gumienny et al., 1999). In
acridine orange (AO) staining experiments, hermaphrodites were placed on
plates to which 500 µl of 100 mM AO was added. After these plates were
incubated for 3-4 hours in the dark, animals were immobilized and viewed by
fluorescent microscopy.
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Results |
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|
In general, protein complexes in which CGH-1 or CAR-1 orthologs have been
found are stabilized by RNA. Interactions among several Drosophila
Me31B complex components are RNA dependent
(Nakamura et al., 2001;
Nakamura et al., 2004
), and
the Pleurodeles waltl (newt) CAR-1 ortholog RAP55 and
Xenopus CGH-1 ortholog Xp54 are each present in RNAse-sensitive
oocyte RNP complexes (Lieb et al.,
1998
). In yeast, mRNA is essential for the formation and
structural integrity of P bodies (Sheth
and Parker, 2003
; Teixiera et al., 2005). We tested whether the
interaction between CGH-1 and CAR-1 also requires RNA, by immunoprecipitating
these endogenous proteins from C. elegans extracts in the presence or
absence of RNase A. Reciprocal western blotting of these immunoprecipitations
showed that the interaction between CGH-1 and CAR-1 was abolished by RNase A
(Fig. 2C), indicating that RNA
is required for CAR-1 and CGH-1 to be present together within a RNP
complex.
Colocalization of CAR-1 and CGH-1
We next investigated whether CAR-1 is distributed similarly to CGH-1 within
the germline and embryo. In adult hermaphrodites, the CAR-1 and CGH-1 proteins
were detectable by western blotting exclusively in the germline
(Fig. 2D), consistent with
previous antibody staining and northern blot evidence that CGH-1 expression is
germline specific (Navarro et al.,
2001). Throughout the gonad CGH-1 associates with P granules, as
revealed by its overlapping localization with the constitutive P granule
component PGL-1 (Fig. 3E-G; not
shown) (Navarro and Blackwell,
2005
; Navarro et al.,
2001
). During the syncytial oogenesis stages
(Fig. 1), P granules are
localized to the perinuclear region, where each is associated with a cluster
of nuclear pores (Pitt et al.,
2000
). After entry into meiosis, many newly synthesized mRNAs
appear to pass through P granules on their way to the central core
(Schisa et al., 2001
). In
parallel, CGH-1 staining levels increase dramatically in response to meiosis
entry, and CGH-1 particles that are independent of P granules then accumulate
within the core (Fig. 3I; not
shown) (Navarro and Blackwell,
2005
; Navarro et al.,
2001
).
Immunostaining of L4 and adult germlines with CAR-1 antisera revealed a
pattern remarkably similar to that of CGH-1
(Fig. 3). CAR-1 levels were
modest in the proliferating stem cells at the distal end of the gonad, then
increased upon entry into meiosis (Fig.
3A). Throughout the gonad some CAR-1 staining was localized to
perinuclear particles, where it overlapped substantially with staining for
CGH-1 and PGL-1 (Fig. 3C-G; not
shown). After germ cells entered meiosis, CAR-1 appeared in cytoplasmic
granules within the syncytial gonad core, in parallel with CGH-1
(Fig. 3A,H-J). Within the core,
most CAR-1 particles colocalized with CGH-1, although some distinct CAR-1 and
CGH-1 foci were also present. The distribution of CGH-1 appeared normal in
car-1(RNAi) animals, but CAR-1 localization was highly abnormal in
cgh-1(RNAi) hermaphrodites (not shown, and K. Oegema, personal
communication) and in the predicted null deletion mutant cgh-1(ok492)
(see Fig. S4 in the supplementary material; Fig. 3K-M). Without CGH-1,
CAR-1 was appropriately associated with P granules in the mitotic region, but
in meiotic cells it was no longer detected at P granules, but accumulated in
large irregularly shaped aggregates within the core
(Fig. 3K-M). By contrast,
antibody staining indicated that PGL-1 localization was not severely disrupted
in cgh-1(ok492) hermaphrodites
(Fig. 3N), as reported
previously for cgh-1(RNAi) animals
(Navarro et al., 2001). Taken
together, the data strongly support the idea that CAR-1 is functionally
associated with CGH-1.
|
CAR-1/CGH-1 association is conserved
Independently of this C. elegans work, sequencing of additional
proteins in the Drosophila Me31B complex
(Nakamura et al., 2001;
Nakamura et al., 2004
)
revealed that its most abundant component is the CAR-1 ortholog Trailerhitch
(Tral) (Table 1; not shown). In
accordance with the interaction observed between CGH-1 and CAR-1
(Fig. 2A,C), endogenous Tral
and Me31B coimmunoprecipitated from a Drosophila ovarian extract, and
was dependent upon RNA (Fig.
5A). To examine the distribution of Tral in Drosophila
ovaries, a strain expressing a GFP-Me31B fusion protein
(Nakamura et al., 2001
) was
immunostained with a Tral-specific antibody
(Fig. 5B-D). Me31B forms
cytoplasmic particles that contain other members of the Me31B complex, along
with specific mRNAs that are translationally regulated by this complex
(Nakamura et al., 2001
;
Nakamura et al., 2004
). Tral
colocalized with GFP-Me31B in these cytoplasmic particles
(Fig. 5B-D). The finding that
Drosophila Me31B and Tral are present in the same complex and
colocalize in the germline suggests that a functional association between
CAR-1 and CGH-1 has been conserved.
car-1 insufficiency increases physiological germ cell apoptosis
To investigate whether CAR-1 and CGH-1 are involved in similar processes,
we assayed whether germline apoptosis is elevated in car-1(RNAi)
adult hermaphrodites. At 24 and 48 hours after the L4 molt, acridine orange
(AO) staining and the counting of germ cell corpses indicated that two- to
threefold more dying germ cells were present in car-1(RNAi) animals
than in wild type (Fig. 6A,B),
similar to the increase seen in cgh-1(RNAi) hermaphrodites
(Navarro et al., 2001). This
cell death required the caspase ortholog ced-3, demonstrating that it
was apoptotic (Fig. 6A).
car-1 RNAi similarly increased the number of corpses present in the
engulfment-defective mutant ced-1(e1735)
(Hedgecock et al., 1983
),
indicating that this increase derived from elevated germ cell death, not
impaired engulfment by the sheath cells (see Table S1 in the supplementary
material).
In C. elegans, physiological germline apoptosis occurs only during
oogenesis and is induced by a specific pathway that does not require either
the p53 ortholog CEP-1 or the proapoptotic protein EGL-1, each of which is
needed for genotoxic stress to induce germ cell death
(Gumienny et al., 1999;
Hofmann et al., 2002
). No
apoptotic cells were detected in car-1(RNAi) males or larval stage
hermaphrodites (not shown), and car-1 knockdown increased germ cell
death comparably in wild type, cep-1 and ced-9(n1950gf)
mutants (Fig. 6A; not shown).
In ced-9(n1950gf) animals, EGL-1 fails to trigger apoptosis, so that
only the physiological germ cell pathway is still active
(Gumienny et al., 1999
;
Schumacher et al., 2005
). We
conclude that CAR-1 and CGH-1, two germline proteins that are present in the
same RNP, are each important for limiting the frequency of germ cell death
through the physiological pathway.
|
Despite their elevated levels of germ cell death, and in contrast to
cgh-1(RNAi) animals (Navarro et
al., 2001), car-1(RNAi) hermaphrodites and males were
fertile. However, car-1(RNAi) hermaphrodites consistently produced
fewer progeny than wild type did (N2, 257.2±15.2, versus
car-1(RNAi), 198.1±34.4; P<0.05;
Fig. 7A). In both wild type and
ced-3(n717) backgrounds essentially all of these car-1(RNAi)
embryos failed to hatch, and instead arrested development with a profound
cytokinesis defect in which cleavage furrows began to form but subsequently
regressed, so that the first embryonic cell division was generally not
completed (Fig. 8)
(Gonczy et al., 2000
;
Piano et al., 2002
).
|
|
|
|
In one-day-old ced-3;car-1(RNAi) animals, the proximal gonad region was filled with a disorganized array of abnormal oocytes that were arrested in diakinesis (Fig. 7B), in striking contrast to either car-1(RNAi) or ced-3 animals (Fig. 6C, Fig. 7C). This accumulation of defective oocytes did not involve accelerated oocyte production, because similar total combined numbers of eggs and oocytes were produced by ced-3 and ced-3;car-1(RNAi) animals during the first 16 hours of adulthood (see Fig. S6 in the supplementary material). When car-1 RNAi was performed in the N2 background, a less severe version of this phenotype eventually appeared in older animals that had ceased to produce progeny (not shown). It is possible that a lack of car-1 leads to the production of individual defective oocytes that are culled by the cell death mechanism, which would thus fulfill a `quality control' function. This model predicts that abnormal car-1(RNAi) oocytes would be generated from the beginning of adulthood. By contrast, during the first 12 hours of adulthood ced-3 and ced-3;car-1(RNAi) animals produced comparable numbers of progeny, and abnormal oocytes began to appear in only a small minority of ced-3;car-1(RNAi) gonads (not shown). It was only as oogenesis continued that ced-3;car-1(RNAi) animals produced progeny at a decreased rate; the animals eventually progressed to gonad failure before sperm were depleted. Taken together, the data suggest that the increased cell death occurring in car-1(RNAi) animals does not involve the elimination of individual defective cells, but instead promotes the production of functional oocytes.
To investigate whether germ cell death similarly facilitates oogenesis in a
context where viable progeny are generated, we examined cpb-3(RNAi)
animals. cpb-3 is expressed primarily during oogenesis
(Luitjens et al., 2000), and
its depletion by RNAi increases the frequency of physiological germ cell death
similarly to cgh-1 or car-1 depletion (not shown)
(Lettre et al., 2004
). In
contrast to car-1(RNAi) animals, cpb-3(RNAi) hermaphrodites
are not only fertile but also give rise to 100% viable progeny
(Fig. 7A, not shown)
(Lettre et al., 2004
).
Interestingly, CPB-3 is the closest C. elegans ortholog of the
RNA-binding CPEB translational regulators
(Luitjens et al., 2000
), which
associate physically and functionally with CGH-1 orthologs in Drosophila,
Xenopus and humans (Table
1) (Mansfield et al.,
2002
; Minshall and Standart,
2004
; Sommerville,
1999
). This last evidence suggests that CPB-3 might be
functionally associated with the C. elegans CGH-1/CAR-1 complex, even
though we did not detect it under our immunoprecipitation conditions.
cpb-3(RNAi) animals produced only slightly fewer progeny than did
wild type, but in the ced-3 background their brood size was
significantly reduced (Fig.
7A). In one-day-old ced-3;cpb-3(RNAi) adults, the
pachytene region of the gonad was abnormally extended towards the proximal end
(Fig. 7E; not shown), a pattern
similar to that observed in old ced-3 animals after sperm depletion
(Fig. 7D; not shown)
(Gumienny et al., 1999). In
addition to this apparent failure to exit pachytene appropriately, in old
ced-3;cpb-3(RNAi) animals abnormal small oocytes that were arrested
in diakinesis accumulated at the proximal gonad end
(Fig. 7F, not shown). These
defects are similar to those seen in ced-3;car-1(RNAi) hermaphrodites
(Fig. 7B). These cpb-3
experiments reveal a second example in which a progressively worsening
oogenesis defect is partially suppressed by an increase in physiological
apoptosis, and suggest that CAR-1 and CPB-3 may function in overlapping
processes.
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Discussion |
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The S. cerevisiae CGH-1 ortholog Dhh1 is a characteristic
component of P bodies and is required for their mRNA degradation function
(Coller and Parker, 2004;
Sheth and Parker, 2003
).
Similarly, the CGH-1 ortholog RCK is typically found in mammalian P bodies
(Cougot et al., 2004
).
Although this suggests that the CAR-1/CGH-1 foci we have described may
correspond to a type of P body, it seems unlikely that mRNA degradation is
their major function during oogenesis, in which mRNAs accumulate. In yeast,
Dhh1-containing P bodies accumulate deadenylated mRNAs and increase in size if
their degradative apparatus is blocked
(Sheth and Parker, 2003
), and
in metazoa deadenylation is the major mechanism for restricting maternal mRNA
translation (Johnstone and Lasko,
2001
). If germline CGH-1/CAR-1 foci are involved in storage or
translational regulation but not degradation of maternal mRNA, it could
explain why they increase in intensity and appear in the gonad core in
parallel with newly synthesized mRNA (Fig.
3A,J) (Gibert et al.,
1984
; Navarro and Blackwell,
2005
; Navarro et al.,
2001
; Schisa et al.,
2001
), and why physical association between CGH-1 orthologs and
the mRNA decapping/degradation machinery has not been detected in the metazoan
germline (Table 1).
In general, RNA helicases are each found in a specific set of RNA-protein
complexes, where they facilitate RNA-RNA or RNA-protein interactions
(Rocak and Linder, 2004). Our
finding that CGH-1 is required for CAR-1 localization specifically after
meiosis entry (Fig. 3K)
suggests that one function of this helicase might be to facilitate the
formation of CGH-1/CAR-1 RNP particles as these proteins and newly synthesized
mRNA accumulate to high levels within the gonad core. In the embryo, CGH-1 and
CAR-1 persist until the approximate 200-cell stage in the germline, but begin
to disappear after the four-cell stage in somatic cells
(Fig. 4), a pattern remarkably
similar to that of a major maternal mRNA subset
(Seydoux and Fire, 1994
). We
speculate that some CGH-1/CAR-1 foci might function as degradative P bodies in
the embryo, where maternal mRNAs must be disposed of in a regulated
fashion.
CAR-1 and CPB-3 are required for normal oogenesis
car-1 is required for oogenesis, as is shown by evidence that, in
car-1(RNAi) hermaphrodites, ACS are produced, physiological apoptosis
is elevated, and brood size is dramatically reduced and abnormal oocytes are
formed if this cell death is prevented
(Fig. 6, Fig. 7A,B). ACS were not
detected in cpb-3(RNAi) or cgh-1(RNAi) animals, although in
the latter case misshapen oocyte fragments may appear in the proximal gonad
(not shown). It is striking that a lack of CAR-1, an apparent RNA-binding
protein, results in two phenotypes that are consistent with cytoskeletal or
membrane abnormalities (ACS production and a cytokinesis defect). In
car-1(RNAi) embryos, maternally derived PAR-1 and PAR-3
(Kemphues et al., 1988) are
appropriately localized to the posterior and anterior, respectively (not
shown), indicating that many aspects of maternal gene expression are intact.
car-1 knockdown therefore does not globally perturb mRNA metabolism
or translation, but affects a more specific cellular process or a mRNA subset.
In Xenopus eggs, the CAR-1 ortholog RAP55 was recently found within a
large RNP that is distinct from the CGH-1/CAR-1 complex, and that is required
for the centrosome-independent pathway of mitotic spindle assembly
(Blower et al., 2005
),
suggesting that CAR-1 might be involved in localizing or regulating specific
mRNAs in multiple contexts in germ cells.
Our evidence that cpb-3 decreases brood size in
ced-3(n717) animals reveals that CPB-3 is important for oogenesis. No
other function has been defined for CPB-3, and RNAi experiments suggest that
it does not function redundantly with the three other C. elegans
CPEB-related proteins (Luitjens et al.,
2000). In some species, CPEB proteins regulate oocyte maturation
through stimulating readenylation and translation of specific mRNAs
(Cao and Richter, 2002
), but
none of the C. elegans CPEB proteins have been implicated in this
process (Lettre et al., 2004
;
Luitjens et al., 2000
).
Interestingly, oocytes are unable to exit pachytene in mice that lack CPEB
(Tay and Richter, 2001
). The
pachytene region is extended in the ced-3; cpb-3(RNAi) hermaphrodite
germline (Fig. 7E), suggesting
that CPB-3 is involved in pachytene exit, and that this function for CPEB
proteins might be conserved in some metazoa.
CGH-1, CAR-1 and physiological apoptosis
Little is understood about the regulation or functions of developmental
germ cell death (see Introduction). In car-1(RNAi) and
cpb-3(RNAi) animals, an increase in cell death partially compensates
for an oogenesis defect, as indicated by the markedly increased severity of
their germline abnormalities in the ced-3 background
(Fig. 7). However, in
ced-3;car-1(RNAi) and ced-3;cpb-3(RNAi) hermaphrodites,
abnormal small oocytes appear only rarely during the first 12 hours of
adulthood (Fig. 7B,F; not
shown), suggesting that these oocytes do not derive from individual abnormal
cells that would otherwise be `culled' by apoptosis. One possibility is that
the consequences for oogenesis of lacking either CAR-1 or CPB-3 are initially
not as severe because germ cell components have been accumulated during larval
stages, but that they become catastrophic after these stores have been
depleted. Physiological apoptosis may then sustain the process of oogenesis by
increasing the supply or facilitating the organization of important
cytoplasmic constituents. It is consistent with this model that the dying
nuclei normally appear to function as nurse cells
(Gumienny et al., 1999), and
that the frequency of physiological apoptosis increases over time in both
wild-type and car-1(RNAi) animals
(Fig. 6A). This cell death
pathway thus may be regulated by a cytoplasmic `checkpoint', which functions
in parallel to the p53-dependent mechanisms that trigger cell death in
response to genotoxic stress.
In C. elegans, whole genome RNAi screening and our experiments
have identified six genes that specifically limit the frequency of
physiological germ cell death (Lettre et
al., 2004; Navarro et al.,
2001
) (this work). These genes encode a predicted E3 ubiquitin
ligase (R05D3.4), a kinase (PMK-3), and four predicted RNA-binding proteins:
CGH-1, CAR-1, CPB-3, and the zinc finger protein T02E1.3a. Although this list
is unlikely to be complete, the small number of genes it includes suggests
that the physiological apoptosis pathway responds to specific cues. It is
remarkable that two of these proteins (CGH-1 and CAR-1) associate with each
other, and that a third (CPB-3) is functionally associated with CGH-1
orthologs in other species (Table
1). This suggests that the regulation of physiological apoptosis
may be influenced specifically by certain functions of the CGH-1/CAR-1
complex. Thus, lack of CGH-1, CAR-1 or CPB-3 may lead to inappropriate
metabolism or regulation of particular mRNAs, resulting in oogenesis
abnormalities that can be compensated for by increased oocyte death. One
intriguing possibility is that the effects of cgh-1 RNAi on
physiological germ cell death might derive from the mislocalization of CAR-1
(Fig. 3K-M). The sterility and
cytokinesis defects seen in cgh-1(RNAi) and car-1(RNAi)
animals, respectively, presumably stem from additional requirements for CGH-1
and CAR-1 function.
In species as diverse as C. elegans and mice, around the time of
pachytene exit it is decided whether each oocyte will survive or die
(Gumienny et al., 1999;
Pepling and Spradling, 2001
).
This process occurs approximately as cytoplasmic communications among oocytes
end. It has been proposed that a function of developmental germ cell apoptosis
is to maintain mitochondrial genome integrity by eliminating unfit
mitochondria (Krakauer and Mira,
1999
; Pepling and Spradling,
2001
). In developing Drosophila oocytes, mitochondria
that are preserved for the germline in the next generation appear to localize
to the Balbiani body (Cox and Spradling,
2003
), an oocyte organelle associated with numerous mRNAs
(Matova et al., 1999
). It is
intriguing that in Xenopus oocytes the CGH-1 ortholog Xp54 is highly
enriched in the Balbiani body, and that, in Drosophila, proteins and
mRNAs that associate with the CGH-1 ortholog Me31B interact transiently with
this structure (Cox and Spradling,
2003
; Smillie and Sommerville,
2002
). These associations suggest the exciting possibility that a
specific connection between the CGH-1/CAR-1 complex and the regulation of
developing germ cell survival may be conserved.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/22/4975/DC1
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