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doi: 10.1242/10.1242/dev.00385
1 Department of Biology, College of William and Mary, Williamsburg, Virginia
23187, USA
2 Laboratory of Biochemistry and Genetics, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda,
Maryland 20892, USA
3 Department of Molecular Genetics, The University of Texas MD Anderson Cancer
Center, Houston, TX, 77030 and Genes and Development Program, Graduate School
of Biomedical Sciences, The University of Texas-Houston, Houston, TX 77030,
USA
Author for correspondence (e-mail:
dcshak{at}wm.edu)
Accepted 9 January 2003
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: mat-1/cdc-27, Asymmetric cell divisions, Meiosis, Cell cycle, APC/C, Caenorhabditis elegans
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Peters, 2002). Like other E3
ubiquitin ligases, the APC/C functions by poly-ubiquitinating its specific
target proteins, thus marking them for destruction by the 26S proteasome.
Models of APC/C function, based largely on studies in unicellular eukaryotes,
suggest that the APC/C acts at two key points during mitosis. Initially, the
APC/C drives cells from metaphase to anaphase by poly-ubiquitinating securin,
the inhibitory binding partner of separase
(Cohen-Fix et al., 1996;
Funabiki et al., 1996b).
Liberated separase can, in turn, proteolytically cleave cohesin proteins
between sister chromatids, thus enabling microtubule-associated forces to pull
the separated sisters to opposing spindle poles
(Uhlmann et al., 1999;
Uhlmann et al., 2000). The
APC/C then promotes mitotic exit by ubiquitinating the M-phase cyclins
(reviewed by Morgan, 1999).
More recently, the APC/C has been shown to function in separating paired
homologs during meiosis I (Cooper et al.,
2000; Davis et al.,
2002; Furuta et al.,
2000; Golden et al.,
2000) and sister chromatids during meiosis II
(Peter et al., 2001;
Taieb et al., 2001). To date,
SnoN, a negative regulator of TGFß signaling
(Stroschein et al., 2001;
Wan et al., 2001) is the only
documented non-cell cycle target of the APC/C. In contrast, SCF, the E3 ligase
that drives the G1 to S transition, has a wide variety of non cell-cycle
targets (reviewed by Deshaies,
1999; Hershko and Ciechanover,
1998).
Nonetheless, the APC/C is likely to have several other targets in addition
to securin and the M-phase cyclins. For instance, the APC/C clearly regulates
spindle and chromosome dynamics beyond disrupting sister chromatid cohesion
since its known spindle-related substrates include Ase1
(Juang et al., 1997;
Visintin et al., 1997)
Aurora-A (Castro et al., 2002;
Taguchi et al., 2002) and
several specialized kinesins (Funabiki and
Murray, 2000; Gordon and Roof,
2001). Recent studies have also implicated developmental roles for
the APC/C during spore formation in yeast
(Asakawa et al., 2001;
Blanco et al., 2001) and
embryonic axis formation in C. elegans
(Rappleye et al., 2002).
However, in the absence of identified developmentally relevant substrates, it
is unclear whether the APC/C regulates these developmental processes directly
or indirectly via its known cell cycle substrates.
Several features make C. elegans an excellent model system not
only for analyzing the role of the APC/C in cell cycle progression but also
for investigating potential links between the cell cycle and development. For
instance, the developmental impact of cell cycle mutants that truncate or
alter the timing of cell lineages can be studied in the context of a known,
invariant cell lineage and well-understood developmental signaling
interactions (reviewed by Lambie,
2002). In fact, the late-developing everted vulva phenotype in
emb-30/apc-4 mutants, and presumably other APC/C mutants, has been
shown to be associated with extended M-phase delays and variable lineage
truncations within the vulva cell lineage
(Furuta et al., 2000). In a
different multicellular context, the C. elegans germline is a highly
proliferative tissue, which like the germline and imaginal discs in
Drosophila, is particularly sensitive to cell cycle defects
(Albertson et al., 1978;
Glover, 1989). Given that the
1000+ cells that compose the mature, syncytial germline arise from just two
cells within hatching L1 larva (Hirsh et
al., 1976), it is not surprising that many APC/C mutants develop
severely reduced germlines due to mitotic defects
(Furuta et al., 2000;
Golden et al., 2000).
Lastly, the powerful combination of excellent cytology, well-established
genetics and RNA interference (RNAi) methodology can be used to analyze how
the APC/C functions at the cellular level to support cell cycle progression
and development of the C. elegans zygote. The one-cell stage of these
large, transparent embryos encompasses both meiotic divisions of the oocyte
chromosomes and the subsequent events leading up to the first mitotic
division. Thus, these zygotes can be used to study common but poorly
understood modifications of the standard cell cycle including how cells
transition between meiosis I (MI) and meiosis II (MII) in the absence of full
M-phase exit (reviewed by Abrieu et al.,
2001), and how cells exit meiosis II and enter pre-mitotic S phase
in the apparent absence of G1 (King et
al., 1994; Vidwans and Su,
2001). In C. elegans, meiotic exit is accompanied by a
striking change in microtubule dynamics. During the meiotic phase, oocyte
chromosomes segregate on small, anastral, acentriolar spindles
(Albertson and Thomson, 1993),
but, upon meiotic exit, the sperm centrosomes begin to nucleate microtubules
as nearby cytoplasmic microtubules disappear
(Clandinin and Mains, 1993).
These developing sperm asters also specify the position of the embryonic
posterior (Goldstein and Hird,
1996; O'Connell et al.,
2000; Sadler and Shakes,
2000; Wallenfang and Seydoux,
2000) and thus microtubules may serve as a common link between
cell cycle progression and the zygote's developmental program.
The role of the APC/C in the various cell cycle and developmental events of
the one-cell C. elegans embryo is just beginning to be elucidated.
When any one of several APC/C subunits is significantly depleted, the affected
embryos experience both a cell cycle block in metaphase of meiosis I and a
corresponding developmental block (Davis
et al., 2002; Furuta et al.,
2000; Golden et al.,
2000; Kitagawa et al.,
2002). To date, this metaphase I block has precluded the analysis
of C. elegans APC/C functions in meiosis II, meiotic exit or the
first mitotic division of the embryo.
Here, we demonstrate that mat-1, a gene known to be involved in
meiosis I metaphase to anaphase transition
(Golden et al., 2000), encodes
the CDC27/APC3 subunit of the APC/C. To analyze late and possibly novel APC/C
functions, we focused on specific hypomorphic defects exhibited by seven
temperature-sensitive mat-1 alleles grown under a variety of
temperature-shift regimes. These studies, in conjunction with RNAi dosage
studies and combinations of APC/C double mutants, demonstrate that different
levels of APC/C activity result in distinct meiotic and post-embryonic
phenotypes. Our analysis not only reveals new roles for the C.
elegans APC/C in meiotic spindle dynamics, meiosis II chromosome
separation, and meiotic exit, but it also extends our understanding of the
male tail defects in APC/C mutants. Importantly, specific APC/C-related cell
cycle defects were found to correlate with predictable and distinct
developmental abnormalities suggesting that at least some of the previously
reported developmental defects (Lyczak et
al., 2002; Rappleye et al.,
2002) might be secondary consequences of meiotic progression and
exit abnormalities.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), while a seventh allele (ye121) was subsequently
isolated in an independent screen for osmotically sensitive mutants
(Rappleye et al., 2002). To
facilitate the analysis of male phenotypes, genetic doubles of mat-1
alleles were constructed with him-5(e1490) (V) or
him-8(e1489) (IV); all male phenotypes were confirmed in
him/+ strains.
Construction of double mutants and GFP lines
Doubly marked mat-1(ax227ts); mat-x or mat-1(ax227ts);
emb-x strains were constructed as follows: unc-38(x20)
mat-1(ax227ts) or mat-1(ax227ts) dpy-5(e61) L4 hermaphrodites
were mated with N2 or him-8(e1489) males. Non-Unc and non-Dpy male
progeny from these crosses were mated with L4 hermaphrodites of the following
genotypes: mat-2(or170ts) unc-4(e120), mat-3(ax148ts) dpy-1(e1),
mat-3(or180ts) dpy-1(e1), emb-27(g48ts) unc-4(e120), and
emb-30(g53ts) dpy-17(e164). Five to ten non-Unc or non-Dpy cross
progeny were picked and from the next generation, the phenotypes of the DpyUnc
double mutant animals were determined. In all cases, double mutant strains
could be identified, though many of the double mutant strains could not be
maintained. All of the above crosses were carried out at the permissive
temperature of 15°C.
For the mat-1 allele ax212ts, double mutants were constructed with an unmarked ax212ts allele. mat-1(ax212ts); him-5(e1490) males were mated with marked L4 hermaphrodites listed above. Non-Unc or non-Dpy F1 progeny were picked, and then 16 F2 Unc or Dpy animals were picked to separate plates (thus homozygous for the second mat or emb loci). One quarter of such animals should also be homozygous for ax212ts. These plates were examined for the presence of candidate double mutants. Again, these experiments were carried out exclusively at the permissive temperature (15°C).
For the mat-1(ye121ts); mat-3(or180ts) double mutant, mat-1(ye121ts); him-8 (e1489) males were mated with mat-3(or180ts) dpy-1(e1) L4 hermaphrodites. From the progeny of non-Dpy F1 animals, 51 F2 Dpy Mat-3 animals were picked to separate plates. One quarter of such animals should also be homozygous for ye121ts. These plates were examined for the presence of candidate double mutants at the permissive temperature. The same cross was performed and F2 embryos (from non-Dpy F1 mothers) were shifted to 25°C. mat-1; mat-3 double mutants were then identified among the Dpy F2 adults.
To construct the mat-1(ax144ts); HIS2B::GFP line, unc-74(x19)
mat-1(ax144ts) heterozygous males were mated into AZ212:
unc-119(ed3); ruIs32 [pAZ132] III. This strain contains an integrated
histone (H2B) GFP transgene expressed under the pie-1 promoter
(Praitis et al., 2001). Unc-74
animals were examined in subsequent generations for GFP expression until a
line segregating all GFP-positive animals was found. This unc-74(x19)
mat-1(ax144ts); ruIs32 line was used for the images in
Fig. 1.
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850 bp fragment of the cDNA clone yk466b3, an EST clone corresponding to
Y110A7A.17. This PCR fragment was used to synthesize dsRNA using the T7
Megascript Transcription kit (Ambion Inc., Austin, TX) according to
manufacturer's instructions. dsRNA was injected into either wild-type animals
(N2 strain) or animals containing the integrated histone H2B::GFP transgene
(strain AZ212) (Praitis et al.,
2001). After a 16- to 20-hour recovery period, gravid AZ212
mothers were directly examined for GFP expression in their embryos. In other
experiments, gravid N2 or AZ212 mothers were dissected and their progeny were
either examined directly for GFP expression or were fixed and stained for
immunofluorescence according to previously published protocols
(Golden et al., 2000). Similar
results were observed when RNAi experiments were performed by standard feeding
protocols (Fraser et al.,
2000; Timmons et al.,
2001) using a construct created by Wallenfang and Seydoux
(Wallenfang and Seydoux,
2000). To observe hypomorphic mat-1 phenotypes
(Fig. 1F), L4 animals were fed
at 15°C and progeny were examined 20-24 hours later. For sep-1
RNAi feeding, the bacterial strain expressing sep-1 dsRNA
(Zipperlen et al., 2001) was
used to feed the C. elegans strain AZ212 at 20°C and progeny were
examined 12-16 hours later.
cDNA synthesis and DNA sequencing
The open reading frame of Y110A7A.17 was confirmed by sequencing the yk10h2
and yk466b3 cDNAs. To obtain the complete 5' UTR sequence, cDNA was
PCR-amplified from wild-type animals using an SL1 5' primer and a primer
within the coding sequence. DNA sequencing revealed a cDNA identical to the
Y110A7A.17 coding region predicted by the C. elegans sequencing
consortium
(http://www.wormbase.org/).
cDNA synthesis and DNA sequencing of Y110A7A.17 from the various
mat-1 alleles was performed essentially as described by Golden et al.
(Golden et al., 2000). The
GenBank accession number for the full-length cDNA is AY081955.
Immunohistochemistry and phenotypic analysis
For immunohistochemical analysis of embryos, germline and somatic tissue,
adult animals were transferred to an 11.5 µl drop of egg buffer
(Edgar, 1995) on a
poly-l-lysine-subbed Color Frost Plus slide (Sigma-Aldrich, Fischer
Scientific). Embryos and gonads were extruded using a 27.5 gauge needle,
covered with a 24x50 mm SuperSlip coverslip (Fischer Scientific), freeze
cracked and processed for antibody staining. For tubulin and phospho-histone
H3 antibody staining, similar protocols and reagents were used as previously
described (Golden et al.,
2000).
The in utero defects of L1 upshifted animals were analyzed either with DIC
optics or epifluorescence (for H2B::GFP transgenic animals) of living adult
animals (Praitis et al., 2001;
Sulston and Horvitz, 1977).
The germline defects of L1 upshifted animals were analyzed by UV
epifluorescence in whole mount, DAPI
(4',6'-diamidino-2-phenylindole)-stained animals that were fixed
with Carnoy II fixative (6:3:1 ethanol/acetic acid/chloroform).
Growth conditions
Animals were grown on a lawn of E. coli (strain OP50) on MYOB
plates (Church et al., 1995).
The analysis of mat-1 male phenotypes was carried out in the mutant
background him-8(e1489), a mutation that increases the frequency of
males by specifically increasing non-disjunction of the X chromosomes
(Hodgkin et al., 1979); but
which does not significantly enhance the mat-1 defects under
restrictive conditions (Table
1). For the analysis of semi-synchronous L1 upshifted males and
hermaphrodites, ten or more mat-1; him-8 hermaphrodites of a given
allele were allowed to lay embryos on MYOB agar plates for 8-12 hours at
16°C before being removed. As soon as 75% of these embryos had hatched,
the plate of largely L1 larvae was shifted to 25°C, and the animals were
subsequently analyzed 42 hours later as young adults. Alternatively,
developmentally arrested L1 larvae were collected from freshly starved plates
and transferred to 25°C plates with food. In late-stage upshift
experiments, animals were semi-synchronized by selecting L4 larvae from
mixed-stage, 15°C populations and shifting them to the indicated
temperature either as L4 larvae or as young adults for a specified
duration.
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To ensure that our analysis of embryos was confined to the mat-1
maternal effects and not its paternal effects
(Golden et al., 2000;
Sadler and Shakes, 2000), the
mat-1 hermaphrodites in these studies were mated to wild-type males.
Importantly, with the exception of ax520 and ax144ts, mat-1
embryos exhibited similar defects regardless of whether their hermaphroditic
parents had been upshifted before, during, or after the L4 larval period, when
C. elegans hermaphrodites generate their full complement of
sperm.
For L4 upshift studies, 10-20 young L4 stage animals that were reared at 15°C were picked onto a fresh plate and shifted to specific temperatures ranging from 20 to 25°C for specified time intervals. For analysis, animals were dissected, fixed and stained for immunohistochemistry. The 15°C and 20°C incubations were done in a Precision Scientific Low Temperature Incubator 815 (±0.3-0.5°C). The 24°C studies were done in an Echotherm (TM) Bench Top Chilling Incubator (±0.01°C). The 25°C studies were done in a Percival Scientific 30 Series Incubator (±0.2°C). The temperatures were monitored with a Barnant RTD (Platinum) Datalogger.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Furuta et al., 2000;
Golden et al., 2000) were
subsequently shown to encode subunits of the APC/C, it seemed likely that
mat-1 also encoded an APC/C related gene. To test this, we searched
the C. elegans sequence database for candidate genes within the
genetic interval known to contain mat-1
(Golden et al., 2000). Within
this region is the single C. elegans homolog of
cdc-27/apc-3. To determine whether this cdc-27/apc-3 homolog
(Y110A7A.17) would produce a metaphase I arrest phenotype, we performed RNAi
with a corresponding EST clone (Fire et
al., 1998; Hunter,
1999). While the uteri of wild-type control hermaphrodites were
filled with rapidly dividing embryos of many different ages
(Fig. 1E,I), the uteri of
hermaphrodites that had been injected with double stranded Ce-cdc-27
RNA were filled with one-cell embryos (Fig.
1G). These cdc-27(RNAi) embryos looked like those from
mat-1 mothers when examined either in utero
(Fig. 1H) or as individual
fixed specimens that had been processed for DAPI staining and tubulin
immunofluorescence (Fig. 1B-D).
The mat-1 and cdc-27(RNAi) embryos accumulated in an
arrested but otherwise normal metaphase I state
(Fig. 1A). In all cases, the
oocyte chromosomes were aligned in a pentagonal metaphase plate on a
barrel-shaped meiotic spindle, and the sperm chromosomes remained highly
condensed. Such embryos also lacked polar bodies (the discarded products of
the meiotic divisions) and had incompletely hardened eggshells.
To confirm that mat-1 encoded the C. elegans cdc-27/apc-3
homolog, the Y110A7A.17 ORF from each of the seven mat-1 mutant
alleles was PCR-amplified and sequenced. The sequencing data revealed unique
missense mutations in the cdc-27 ORF for each mat-1 mutant
allele (Fig. 2), indicating
that mat-1 does indeed encode the C. elegans cdc-27 homolog.
The seven mutations are scattered throughout the coding sequence; five
mutations alter highly conserved residues while those in ax520 and
ye121 lie immediately adjacent to highly conserved regions. None of
the mutations affect potential phosphorylation sites, but five occur within
the conserved tetratricopeptide repeats (TPR) that are thought to mediate
protein-protein interactions in a wide variety of proteins including three
other APC/C components (Lamb et al.,
1995).
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)
(ye121, this study). This metaphase I-arrest phenotype is identical
to the most extreme RNAi phenotype of any APC/C subunit
(Davis et al., 2002;
Furuta et al., 2000;
Golden et al., 2000) and is
likely to reflect the earliest function of the APC/C. However because this
early block precludes the analysis of later functions, we reasoned that
additional functions of the APC/C in either M-phase exit and/or early
embryonic development could be studied in temperature-sensitive (ts)
mat-1 hermaphrodites raised at various intermediate temperatures. In
these experiments, L1 larvae were shifted to various temperatures and their
resulting clutches were scored for overall embryonic lethality and whether the
affected embryos were dying as meiotic one-cell stage or multicellular embryos
(Table 1). Multicellular
embryonic lethality was previously reported in a subset of what are now known
to be APC/C mutant alleles (Golden et al.,
2000; Rappleye et al.,
2002), but our finding that multicellular embryonic lethality is
associated with all seven mat-1 alleles at intermediate temperatures
(Tables 1 and
2), confirms this as a general,
rather than an allele-specific, phenotype. Similar phenotypes were observed
when the levels of maternal mat-1 mRNA were directly lowered by RNAi
(Fig. 1F). When fed bacteria
expressing mat-1/cdc-27 dsRNA, affected wild-type hermaphrodites
initially produced viable offspring, but subsequently produced a brief burst
of multicellular dead embryos (20-24 hours) before producing only one-cell
arrested embryos. These results, along with the observation of similar
phenotypes in other temperature-sensitive APC/C mutants
(Golden et al., 2000;
Rappleye et al., 2002),
suggest that this phenotype is associated with intermediate levels of APC/C
activity rather than the specific disruption of either MAT-1 or a particular
MAT-1 subdomain.
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), this result suggested that normal embryonic
development can tolerate minor (20-40 minute) delays in meiotic progression.
Under semi-permissive conditions, mat-1 hermaphrodites accumulated
3-6 times as many meiotic embryos as same-temperature controls (green in
Table 1; Fig. 1J,K) and, under fully
restrictive conditions, mat-1 hermaphrodites produced only
meiotically arrested one-cell embryos (MeOC; red in
Table 1;
Fig. 1L). Ovulation rates were
unaffected and could not account for the observed accumulation of meiotic
embryos (data not shown). To test whether these meiotic embryos were accumulating specifically in meiosis I or, alternatively, at various stages throughout meiosis, control and mutant embryos were dissected from wild-type or mutant mothers and prepared for DNA (DAPI) staining and anti-tubulin immunofluorescence. Embryos isolated from the uteri of wild-type mothers contained approximately equal numbers of meiosis I (MI) and meiosis II (MII) embryos indicating that these stages are normally of equal duration (Table 2). Under permissive conditions, mat-1 hermaphrodites contained more meiotic embryos than wild-type controls (blue in Table 1), but roughly equal numbers of MI and MII embryos (Table 2). However, increasingly restrictive temperatures were associated with an increasing skew in the MI:MII ratios (green in Tables 1 and 2), and, at fully restrictive temperatures, only MI arrested embryos were observed.
Although semi-permissive temperatures were associated with significant mother to mother phenotypic variability (Table 1 and 2), inspection of the overall MI:MII ratios and polar body numbers revealed two phenotypic subgroups amongst multiple mat-1 alleles. Less affected mothers produced MI, MII, and multicellular embryos, but owing to an extended MI, the MI:MII ratio was 3:1. Older embryos within such mothers had two positionally distinct polar bodies (2PB), indicating that both meiotic divisions had occurred. More severely affected mothers produced only MI and multicellular embryos. Whether examined by DAPI staining or DIC optics, these older embryos had only a single polar body (1PB), suggesting that they had exited meiosis after completing only a single meiotic division. Likewise, pronuclear stage embryos lacked the extra maternal pronucleus that would be expected if a second meiotic division had occurred in the absence of cytokinesis. Under conditions favoring the production of 1PB embryos, a few mothers within the population produce MII embryos, resulting in an overall MI:MII ratio of 9:1 (Table 2), however such variation was not observed amongst sibling embryos from the same mother. For clarity, we will hereafter refer to individual embryos as belonging to the `1PB class' or `2PB class'.
Multicellular embryonic lethality is associated with severe meiotic
defects
In contrast to previous reports
(Rappleye et al., 2002),
hypomorphic mat-1 embryos proved to exhibit striking meiotic defects.
In wild-type C. elegans embryos, there are four basic stages to each
meiotic division (Fig. 3A-D).
In mature MI oocytes, nuclear envelope breakdown occurs just prior to
fertilization. Fertilization is followed by the rapid assembly of an anastral
meiotic spindle and the congression and alignment of paired oocyte homologs on
a metaphase plate (Fig. 3A).
During early anaphase I (Fig.
3B) the homologs separate as the spindle shortens and then rotates
to a position perpendicular to the cortex
(Albertson, 1984). By late
anaphase I (Fig. 3C), a
prominent microtubule bundle lies almost entirely between the separated
homologs and possesses a morphology reminiscent of a cinched haystack. During
telophase I (Fig. 3D) the
individual chromosomes coalesce into two opposing chromatin masses as the
spindle splits during the highly asymmetric cell division that forms the first
polar body. This sequence is reiterated during MII but is immediately followed
by nuclear envelope formation and a round of DNA synthesis. In the absence of
a functional APC/C, embryos remained blocked in metaphase I; with time, the
meiotic spindle disassembles but nuclear envelopes never reassemble
(Golden et al., 2000).
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).
Consistent with meiotic chromosome segregation defects, the maternal pronuclei
of early post-meiotic embryos also were frequently misshapen (data not shown).
Similar defects were observed in both meiotic divisions of 2PB class embryos.
Analysis of MII embryos revealed relatively normal metaphase figures
(Fig. 3I,I'), yet MII
spindle rotation and second polar body formation occurred in the absence of
sister chromatid separation (Fig.
3J), resulting in a MII `cut' phenotype. In addition, mutant
spindles were frequently longer than wild type
(Fig. 3K'). These MII
defects suggest a role for the C. elegans APC/C in either spindle
shortening or MII, although our data do not preclude the possibility that many
or all of these MII defects are a secondary consequence of MI abnormalities.
Importantly, we identified no semi-permissive temperature conditions that
resulted in either metaphase II arrest or an accumulation of MII embryos.
Specific meiotic defects correlate with specific developmental
consequences
In wild-type C. elegans embryos, there is a predictable and tight
correlation between the developmental and cell cycle events of the one-cell
stage (Sadler and Shakes,
2000). Key developmental events include the formation of an
impermeable, three layer eggshell during the late meiotic phase
(Chitwood and Chitwood, 1974)
and the post-meiotic establishment of the embryo's anterior-posterior (AP)
axis (for reviews, see Gotta and Ahringer,
2001; Lyczak et al.,
2002; Pellettieri and Seydoux,
2002). Importantly, AP polarization appears to be triggered, in
part, by growth of the sperm-derived microtubule asters
(O'Connell et al., 2000;
Sadler and Shakes, 2000;
Wallenfang and Seydoux, 2000).
AP polarization culminates in a highly asymmetric first mitotic cell division
that results in the formation of sister blastomeres that differ in size,
synchrony, and the orientation of their cell divisions.
Metaphase I arrested mat-1/cdc-27 (RNAi) embryos fail to
develop either an impermeable eggshell or a stable A-P axis, indicating that
the early cell cycle block is coupled with a corresponding developmental block
(Golden et al., 2000;
Wallenfang and Seydoux, 2000)
(this study). To determine the effect of a partial APC/C depletion, embryos
from mat-1 mothers grown under semi-permissive conditions were
analyzed for eggshell and polarization defects. Mutant embryos with two
distinct polar bodies made eggshells that were impermeable to the lipid
soluble DNA dye Hoechst 33248 whereas mutant embryos with a single polar body
made Hoechst-permeable eggshells. To assess potential polarization defects,
dividing sister blastomeres of either mutant or wild-type two-cell embryos
were scored for their relative size, synchrony and spindle orientations
(Table 3, Fig. 4). Compared to wild-type
controls, hypomorphic mat-1 embryos either exhibited increased
variability in the positioning of their first cleavage plane
(Table 3) or, under
increasingly stringent conditions, divided symmetrically. Symmetric divisions
were strongly correlated with the presence of a single polar body (14/14;
Fig. 4N,O). In general, sister
blastomeres of such 1PB class embryos divided synchronously with both spindles
oriented perpendicular to the long axis of the embryo
(Fig. 4N,O). In contrast, those
of 2PB class embryos were variably intermediate in their relative size,
synchrony, and cleavage orientations (15/15;
Fig. 4I,J). Lastly, although
germline-specific P-granules segregated normally in most 2PB class embryos,
P-granule segregation failed in both MI-arrested embryos and in symmetrically
and synchronously dividing 1PB class embryos (data not shown). In summary, 1PB
class embryos exhibited consistently more severe defects in the symmetry,
timing, and cleavage orientations of their early cell divisions when compared
with either 2PB class embryos or wild-type controls.
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). Such flows were absent in 1PB mutant embryos, and in
15/15 same-stage embryos, the male pronuclei failed to relocate from their
initial cortical position to one of the two embryonic ends
(Fig. 4K). In addition, both
the expansion and separation of the sperm asters was delayed
(Fig. 4L,M).
Displaced male pronuclei have also been reported in embryos partially
depleted of separase, the key effector of metaphase APC/C activity
(Rappleye et al., 2002). Our
own observations revealed that wild-type mothers fed for a specific short time
on sep-1 dsRNA-producing bacteria produce embryos that specifically
fail to relocate their male pronuclei. However, in striking contrast to the
earlier report, such defects were always associated with severe meiotic
defects, namely the presence of a single polar body (12/12;
Fig. 5).
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). In one-cell C. elegans embryos, exit out of MII is
complex because it involves both a direct M to S transition (no apparent G1)
and the final conversion of the fertilized oocyte into a zygote. This M to S
phase transition is marked by several events including the disappearance of
M-phase cell-cycle markers such as phosphorylated histone H3 (p-H3)
(Fig. 6), the extensive
remodeling of the sperm chromatin, the disassembly of the meiotic spindle, and
the duplication and nucleation of the paternally derived centrosomes
(Albertson and Thomson, 1993;
Sadler and Shakes, 2000).
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Analysis of M-phase exit in 1PB class embryos revealed several striking abnormalities. In particular, the post-meiotic maternal chromosomes continued to stain with p-H3 antibodies, albeit unevenly, even after nuclear envelope reformation (10/10; Fig. 6Q). In addition, the male pronucleus sometimes stained aberrantly with p-H3 (5/10; Fig. 6R) even after centrosome duplication and the initiation of aster formation (Fig. 6N). In addition, such embryos frequently contained large, disorganized meiotic spindle remnants that, unlike their wild-type counterparts, appeared to extend into the cortical interior (Fig. 6M). Such defects suggest that the aberrant meiotic exit of the 1PB class directly out of MI disrupts the normal coordination of the various cellular events that accompany the process of meiotic exit and that set the stage for the proper development of the zygote.
Analysis of APC/C double mutants reveal latent mitotic defects in
apparently meiotic-specific alleles
In previous studies, a subset of temperature-sensitive mat alleles
were found to exhibit defects not only in the meiotic divisions of the
post-fertilization embryo but also in the mitotically proliferating germline,
male tail and hermaphrodite vulva (Furuta
et al., 2000; Golden et al.,
2000). Amongst mat-1 alleles, only two alleles
(ax144 and ax520) proved to result in significant germline
proliferation defects (Golden et al.,
2000) (Table 4;
Fig. 7) and only three alleles
(ax144, ax520 and ax212) produced significant defects in the
male tail and hermaphrodite vulva (Table
4; Fig. 8). In
contrast, other alleles either resulted in no mitotic defects or moderate
defects in only a subset of the population
(Table 4). Although such
results could suggest that the other mat-1 alleles were meiotic
specific, the discovery that the mat-1 molecular lesions were
scattered throughout the protein (Fig.
2) suggested that the meiotic divisions of the oocyte may simply
be more sensitive to a partial loss of APC/C function. If the apparent
allele-specificity was merely a matter of dosage, additional mat-1
alleles might be expected to display mitotic defects in double mutant
combinations with other APC/C genes.
|
|
|
). At 15°C, all of the double mutants developed to
adulthood, but some of these double mutant mothers produced clutches of dead
embryos (Table 5) suggesting
that, even at 15°C, APC/C activity in mat-1(ax227) and
mat-1(ax212) animals is not completely normal. To specifically test
whether these double mutant combinations would exhibit `latent' germline
defects, ax227 double mutants from heterozygous parents were shifted
to the restrictive temperature as embryos or L1 larvae. While all of the
double mutants developed through the adult stage, many were sterile
(Table 5). Likewise,
mat-1(ye121); mat-3(or180) doubles were also sterile at 25°C.
Taken together, these results indicate that the molecular lesions in
ax212, ax227 and ye121 are not meiotic-specific, even
though, as single mutants, these alleles provide sufficient APC/C function to
support normal germline proliferation. Although we did not observe enhancement
of male tail and hermaphrodite vulva defects in these mat-1 double
mutant experiments, these somatic mitotic defects were enhanced in the double
APC/C Mel combination of emb-27(g48) and emb-30(g53) (P.S.,
unpublished).
|
;
Golden et al., 2000) as were
everted vulva (Evl) defects in emb-30/apc-4 mutants
(Furuta et al., 2000). For
mat-1 Evl phenotype, see Fig.
8I,J. However, no such direct link had been made between the male
tail phenotype and cell cycle defects. To test whether the mat-1 male
tail defects (Fig. 8A-D) also
correlated with mitotic cell cycle defects, L1 larvae from either the severely
affected ax144; him-8 strain or him-8 controls were shifted
to 25.5°C for periods ranging from 18 to 36 hours. The animals were
dissected to separate the gonad from the soma, and then processed for
immunostaining with anti-p-H3 antibody. The somatic staining patterns in the
ax144 samples were indistinguishable from the controls during the 18-
and 24-hour time points. However, a dramatic excess of p-H3 staining cells was
found in the posterior of ax144 L4 male larvae collected 30 to 36
hours (L4) after upshift (Fig.
8H,K) when compared to the wild-type controls
(Fig. 8F,K). Importantly, only
some of these p-H3-positive cells were in metaphase, suggesting that the APC/C
defect is prolonging more than just the metaphase to anaphase transition.
Because both the final cell divisions and morphogenetic events of male tail
formation occur during this same L4 period, it is likely that at least some of
these p-H3-positive cells were progenitors of male tail cells. Thus, as in the
vulva, the morphological defects in the mutant male tails are likely to stem
from a combination of M-phase delays and potential truncations within these
late-dividing cell lineages. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the present
study, we have employed C. elegans temperature-sensitive mutants in
the APC/C subunit MAT-1/CDC-27 to reveal a wide range of defects at both the
organ and cellular level that can result from partial depletions of APC/C
activity. When MAT-1 was significantly depleted by either shifting
mat-1(ts) mothers to 25.5°C or injecting wild-type mothers with
double-stranded mat-1/cdc-27 RNA, the affected oocytes were
fertilized but arrested as one-cell embryos with their paired homologs locked
in metaphase of meiosis I. In contrast, when various temperature shift regimes
were used to create a graded MAT-1 depletion series, the resulting one-cell
embryos exhibited an extended meiotic phase as well as several
dosage-dependent meiotic defects ranging from incomplete chromosome separation
and abnormal meiotic spindle dynamics to a complete bypass of the second
meiotic division. While metaphase I-arrested mat-1 embryos exhibit a
corresponding developmental block at the meiotic one-cell stage, hypomorphic
mat-1 embryos exhibited dosage-dependent meiotic defects that
correlated with specific defects in early development
(Fig. 9). These developmental
defects included altered pronuclear migration patterns, aberrant polarization,
and alterations in the normal timing and orientation of the embryo's mitotic
cell divisions. When mat-1 mutants were upshifted as L1 larvae,
additional somatic and germline defects were observed in three of the seven
alleles; other alleles could be induced to exhibit these phenotypes in double
mutant combinations with other APC/C subunits.
|
), suggest that all of these alleles, except ax72 and
ax520, are temperature sensitive for protein folding. In such
mutants, high temperatures destabilize an intermediate in the folding pathway
without altering the functions of the folded state
(King et al., 1996). None of
the seven mat-1 alleles are molecular nulls, however, we believe that
the mat-1 metaphase I arrest phenotype reflects an absence of APC/C
function within the oocyte since RNAi embryos of seven different APC/C
subunits display an identical phenotype (this study)
(Davis et al., 2002). Contrary
to our initial expectations, none of these mat-1 alleles, except
perhaps ax72, proved to be meiotic specific. Perhaps mutations in TPR
domains are poor candidates as single function lesions if the TPR domains in
MAT-1/CDC-27 serve primarily to glue the complex together. Theoretically,
mutations of phosphorylation sites might cause single function defects, but no
such lesions are represented within our current mutant collection.
Tissue specificity of C. elegans cell cycle mutants
Although none of our mat-1 alleles are molecular nulls, homozygous
null embryos of other APC/C subunits survive embryogenesis and develop into
sterile hermaphrodites with everted vulva [EMB-30/APC-4
(Furuta et al., 2000),
MAT-2/APC-1 (Davis et al.,
2002), MAT-3/CDC-23 (D. Garbe and M. Sundaram, personal
communication), and APC-11 (A.G., unpublished)]. Furthermore, similar
phenotypes have been described for null mutants of other cell cycle genes
including cyclin D (Boxem and van den
Heuvel, 2001; Park and Krause,
1999) cyclin E (Fay and Han,
2000) CDK-4 (Boxem and van den
Heuvel, 2001; Park and Krause,
1999) and CDK-1 (Boxem et al.,
1999). Presumably these cell cycle mutants survive embryogenesis
and larval development because of high levels of persistent maternal mRNA or
protein stores, and, in the case of temperature-sensitive mutants, the
resistance of previously synthesized and complexed proteins to unfold at
restrictive temperatures. The biology of C. elegans may also explain
the surprisingly mild phenotypic defects. Since C. elegans embryos
develop in the absence of net growth, maternal stores of cell cycle proteins
may persist at sufficient concentrations to support cell divisions throughout
embryogenesis. Likewise, surprisingly normal larval development can occur in
the complete absence of larval cell divisions, albeit the resulting adults are
sterile and uncoordinated (Albertson et
al., 1978). Similarly, many Drosophila cell cycle mutants
(Gatti and Baker, 1989),
including those in APC5 (Bentley et al.,
2002) survive embryogenesis and larval development before dying
during the prepupal stage with underdeveloped imaginal discs.
The germline, vulva, and male tail may be particularly sensitive to defects
in the APC/C and other cell cycle genes because all three are generated
through mitotic proliferation of embryonic blast cells, with the majority of
divisions occurring during the last two larval stages. In the case of the
germline, two germline progenitor cells proliferate more than any other blast
cells in the hatching L1 larvae. The cell lineages giving rise to the
developing male tail and hermaphrodite vulva do not involve more cell
divisions than those giving rise to other somatic structures, but these
divisions are sufficiently late that maternal stores may be inadequate. In
addition, proper development of these organs requires a high degree of
coordinated signaling interactions
(Lambie, 2002) and thus may be
particularly sensitive to M-phase delays and/or abnormalities that result in
either truncation or altered timing of the cell lineages. Interestingly,
mat-1 mutants also exhibit gonad migration defects at low frequency
(M.A. and D.S., unpublished), which may stem from cell signaling defects
during gonadal development. Taken together, these studies suggest that, in
multicellular organisms, the sensitivity of a particular tissue to hypomorphic
APC/C levels will depend on total cell proliferation and the degree to which
coordinated cell division is linked to morphogenesis.
Hypomorphic mutants reveal late and novel functions for the
APC/C
APC/C dosage studies revealed a hierarchy of APC/C functions. Upon partial
depletion of APC/C levels, chromosome separation is affected more than spindle
shortening, and spindle shortening is more affected than either spindle
rotation or polar body formation. A similar hierarchy has been observed in
fission yeast; APC/C null mutants arrest in metaphase whereas most ts mutants
exhibit a `cut' phenotype in which cytokinesis proceeds in the absence of
chromosome segregation (Chang et al.,
2001). To date, the basis for this differential sensitivity
remains unclear. In other systems, APC/C substrate specificity is regulated by
its associated WD repeat proteins. Current models suggest that securin is
targeted primarily by APCCdc20
(Cohen-Fix et al., 1996;
Funabiki et al., 1996b)
whereas cyclin B is targeted primarily by APCCdh1, although recent
studies suggest that the specific roles of these WD proteins may be more
variable (Hsu et al., 2002;
Sigrist and Lehner, 1997;
Stegmeier et al., 2002;
Yamaguchi et al., 2000;
Yeong et al., 2000). Recent
studies also indicate that APCCdc20/Fizzy is the sole form of the
APC/C during C. elegans meiosis; fzy-1(RNAi) embryos arrest
in metaphase I (Kitagawa et al.,
2002) whereas cdh-1/fzr-1(RNAi) embryos develop into
sterile adults (Fay et al.,
2002). If the CDH1/FZR-1 ortholog does not, in fact, function in
meiotic C. elegans embryos, the observed hierarchy may reflect a
differential affinity of APCCdc20 for its various targets.
Alternatively, if C. elegans has additional, yet undiscovered meiotic
specificity factors (WD repeat proteins), perhaps only the higher affinity
complexes function at low APC/C levels.
Our studies have also revealed a potentially novel APC/C function in late
anaphase meiotic spindle shortening. In mitotic cells, APC/C contributes to
changes in anaphase spindle morphology in part by ubiquitinating
Ase-1 (Juang et al.,
1997; Visintin et al.,
1997) and various kinesins
(Gordon and Roof, 2001).
Whether meiotic spindle shortening is directed through the same or unique
APC/C substrates will be the subject of future studies.
APC/C may not be required for meiosis II exit
One of the more intriguing findings of this study was the differential
effect that APC/C depletions had on the two meiotic divisions
(Fig. 9). While partial
depletions of APC/C activity disrupt the separation of paired homologs in MI
and sister chromatids in MII, extended delays and/or arrest of the metaphase
to anaphase transition occurred only during MI. While both the 1PB and 2PB
class embryos experience extended MI delays, it was surprising that the MII
chromosome separation defects in 2PB class embryos were never coupled with a
metaphase II arrest or a prolonged MII cell cycle. One explanation for this
result is that meiosis I is as sensitive, or more sensitive, than meiosis II
to decreased APC/C activity, and thus it may not be possible experimentally to
observe a MII arrest (since such temperatures result in a MI arrest).
Alternatively, the differential impact reflects true differences between the
two meiotic divisions. More specifically, oocytes normally transition between
MI and MII without fully exiting M-phase; whereas exit from MII absolutely
requires the destruction of M-phase cyclins and is accompanied by nuclear
envelope reformation (Kobayashi et al.,
1991; Minshull et al.,
1991). While mitotically dividing cells drive M-phase with
APCCdh1 and the G1 to S transition with SCF
(Deshaies, 1999), one-cell
C. elegans embryos apparently lack a G1 stage and thus the final
stages of MII exit in C. elegans and potentially other embryos could
be driven not by the APC/C, but rather by SCF or another cullin/RING finger
complex. Consistent with this model, RNAi depletion of a CUL-2-containing
complex results in severe M
