The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants “mat” for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog.
Eukaryotic cell division is a highly regulated process during which cells duplicate their chromosomes, align them at a metaphase plate, and then segregate them equally to each of their two daughter cells. Accurate chromosome segregation during the metaphase to anaphase transition is essential for proper cell function, since errors in chromosome segregation can lead to cell death, sterility, birth defects, or malignant cancers. Not surprisingly, genetic studies of yeast mitosis suggest that the transition out of metaphase and through anaphase is highly regulated both in terms of the sequence of events and the existence of checkpoint regulators. Driving the process forward is a multiprotein E3–ubiquitin ligase complex known as the anaphase promoting complex or cyclosome (APC/C) that functions to sequentially target key cellular components for proteolytic destruction (Zachariae and Nasmyth 1999). APC/C initiates the metaphase to anaphase transition by first targeting the destruction of the anaphase inhibitor Pds1/Cut2 (Yamamoto et al. 1996) whose absence permits the separation of sister chromatids (Ciosk et al. 1998). APC/C then drives the process forward as it subsequently targets Ase1 and cyclin B for destruction (Peters 1999). Ase1 destruction permits spindle elongation during anaphase B (Juang et al. 1997), and cyclin B destruction permits M phase exit.
Cells must avoid premature activation of APC/C and precocious exit from metaphase, because once chromosomes separate during anaphase, defects stemming from misaligned or stray chromosomes can no longer be corrected. The idea that cells might have a metaphase checkpoint (also called spindle assembly checkpoint or kinetochore attachment checkpoint) was first suggested by the observation that cells treated with microtubule inhibitors arrest in a metaphase-like state (Eigsti and Dustin 1957). Recently, this arrest has been shown to be mediated by a set of checkpoint proteins that specifically block the activity of the APC/C in the presence of either abnormal spindles or unattached kinetochores (for review see Gardner and Burke 2000).
A combination of genetic, cytological, and biochemical studies has elucidated many of the molecular mechanisms that underlie these critical cell cycle controls (Murray and Hunt 1993; Nasmyth et al. 2000). These studies reveal that the same basic mechanisms drive the processes of cell division and chromosome separation in both yeast and animal cells, and that many key players are highly conserved in their molecular sequences. Nevertheless, given that many cells differ in the specific details of their cell division processes, underlying mechanistic differences are also likely to exist. For instance, animal cells disassemble their nuclear envelopes before chromosome separation, but yeasts do not. Since the mitotic proteins of yeasts and animal cells function within distinctive cellular environments, they may also be regulated differently. Likewise, the mechanisms that separate sister chromatids during mitosis and meiosis II may differ from those that separate chiasmatically linked homologous chromosomes during meiosis I. Although the loss of sister chromatid cohesion is critical for all of these divisions, centromeric cohesion is specifically protected only during meiosis I (Miyazaki and Orr-Weaver 1994; van Heemst and Heyting 2000). On the other hand, both the APC/C target, Pds1, and the APC/C regulator, MAD2, have been recently shown to regulate the separation of not only sister chromatids, but also homologous chromosomes (Salah and Nasmyth 2000; Shonn et al. 2000). Another largely unexplored area of investigation is whether APC/C-related proteins and their substrates function differently during oocyte and spermatocyte meiosis, given that oocyte meiosis occurs on an acentriolar spindle, whereas sperm meiosis employs a centriole-based spindle.
Here, we present the isolation and analysis of a collection of temperature-sensitive (ts) mutants in the nematode Caenorhabditis elegans whose defects specifically block the metaphase to anaphase transition during the germline mitotic and meiotic divisions. Furthermore, we show that at least one of these genes encodes a subunit of the APC/C. This mutant collection provides an important addition to the analysis of the metaphase to anaphase transition for several reasons. First, because the alleles are ts, they can be used to analyze the role of essential genes during gametogenesis. Such analysis would not be possible in null mutants of genes that are required for the early mitotic divisions of either the soma or the developing germline. Second, because the germline is the only tissue in C. elegans adults that continues to undergo cell divisions, adult upshift experiments allow germline-specific defects to be studied in the absence of complicating somatic defects. Third, these germline cell divisions are both interesting and distinct for the following reasons. (a) A unique aspect of meiosis I is that the paired homologues are linked by chiasmata. (b) Oocyte and spermatocyte meiosis differ drastically in their associated spindle structures and cytokinesis patterns. (c) Mitotically dividing germ cell nuclei exhibit an intriguing cell cycle independence despite the fact that they are cytoplasmically linked (Hirsh et al. 1976). (d) Finally, because the meiotic germ cells are arranged in temporal order along the distal–proximal axis of the adult gonad, sequential stages of meiosis can be easily observed. With a focus on the metaphase to anaphase transition, this collection of ts mutants provides a unique opportunity to compare the phenotypic consequences of M phase defects during three different types of cell divisions: oocyte meiosis, spermatocyte meiosis, and germline mitosis.
Materials and Methods
Genetic Screen for Ts Embryonic Lethal Mutants
Ts embryonic lethal mutants were isolated in two separate genetic screens, both modified from Kemphues et al. 1988. All steps were carried out at 15°C, except where noted. The ax alleles were isolated as follows: L4 hermaphrodites of the genotype him-3 (e1147) IV; lin-2 (e1309) axIs36 X were mutagenized with 25 mM ethyl methanesulfonate using standard procedures (Brenner 1974). lin-2 mutant hermaphrodites lack a functional vulva, and, thus, retain most of their progeny in their uteri. axIs36 is a chromosomally integrated transgene containing a pes-10:GFP fusion that was used in a secondary screen not described here (Wallenfang, M. and G. Seydoux, unpublished results). F2 animals, synchronized by hypochlorite treatment of F1 gravid adults (Emmons et al. 1979), were shifted as L4 larvae to 25°C for 20 h and back down to 15°C for an additional 20 h. F2 adults containing dead embryos were transferred individually to new plates at 15°C. 3 d later, plates were examined for the presence of viable F3 progeny, indicating an embryonic lethal mutant was rescued by the shift to the permissive temperature. From ∼900,000 mutagenized genomes, 1,197 ts embryonic lethal mutants were isolated. With the protocol used, most, but perhaps not all, of these alleles are independent. The terminal phenotypes of these mutants was determined in a secondary screen by shifting L4 hermaphrodites to 25°C for 12–16 h and viewing the accumulated in utero embryos under DIC (differential interference contrast) optics on a Zeiss Axioplan II compound microscope (ZEISS). From 1,197 ts embryonic lethal mutants, we recovered 30 mutants that arrest at the one-cell stage. Five of these one-cell arrested mutants have cytokinesis defects and will not be described here. The remaining 25 ax alleles are the subject of this report.
The or alleles described here were isolated in a similar screen, except for the following differences: the starting lin-2 strain lacked him mutations and integrated transgenes, ethyl methanesulfonate was used at 20 mM, and F2 animals were upshifted as L4 larvae for 25–30 h. For screening, F2 animals were suspended in M9 buffer, and bloated animals containing dead embryos were transferred individually to new plates at 15°C. From ∼1,000,000 mutagenized genomes, ∼300 ts embryonic lethal mutants were isolated. Of these, 14 mutants arrested at the one-cell stage. Seven of these are described here, and the remaining seven have unrelated cytokinesis defects (Encalada et al. 2000).
Strains and Alleles
The Bristol strain N2 was used as the standard wild-type strain. The marker mutations, deficiencies, and balancer chromosomes used are listed by chromosome as follows: LGI: dpy-5(e61), unc-13(e450), bli-4(e937), dpy-14(e188), unc-74(x19), unc-38(x20), unc-63(x18), hDf6, sDf4, hDp31(I;f); LGII: rol-1(e91), unc-4(e120), bli-1(e769), unc-53(e404), mnDf90, mnDf86, mnDf77, mnC1 (dpy-10[e128] unc-52[e444]); LGIII: unc-32(e189), dpy-19(e1259), glp-1(q339), daf-7(e1372ts), dpy-1(e1), unc-36(e251), tDf9, mnDp37 (III;f); LGIV: him-3(e1147), him-8(e1489); LGV: him-5(e1490); and LGX: lin-2(e1309). Culturing, handling, and genetic manipulations of C. elegans were performed using standard procedures (Brenner 1974).
Newly isolated mutations were outcrossed three to five times, and complementation was tested to one another until map positions were established. Linkage analysis, deficiency complementation, two factor mapping, and three factor mapping were used to map the three mat loci and the new alleles of emb-27 and emb-30. Additional emb-27 and emb-30 map data was reported previously (Cassada et al. 1981; Sigurdson et al. 1984; Furuta et al. 2000). Our mapping crosses place mat-1 on LGI close to, or left of, unc-63. Deficiency crosses place mat-2 on LGII between the left breakpoint of mnDf87 and the right breakpoint of mnDf90, but not under either mnDf86 or mnDf87. mat-3 maps left of dpy-1 and daf-7 on the far left arm of LGIII (see Fig. 2). Data from these crosses are available from the C. elegans database WormBase (http://www.wormbase.org). Because mat-1(ax72) and mat-1(ax520) homozygotes were difficult to maintain at 15°C, these alleles were maintained as heterozygotes over an unc-38(x20) dpy-5(e61) chromosome. Likewise, emb-27(ax189) was maintained over the mnC1 balancer chromosome. Because mat-1(ax212) is leaky at 25°C, it was characterized at 26°C. To facilitate complementation tests and the analysis of male phenotypes, genetic doubles of various mat alleles with either him-5(V) or him-8(IV) were constructed; all male and spermatogenesis phenotypes were secondarily confirmed in him/+ strains.
Mutant stocks were maintained at either 15 or 16°C. For the two-cell embryo shift-up experiments, adult hermaphrodites were raised at 15°C, and then dissected in a drop of egg salts (Edgar 1995), using 27.5 gauge needles. Isolated two-cell embryos were transferred to prewarmed 25.5°C plates for further development. In L1 upshift experiments, threefold embryos were picked to fresh plates at 15°C and shifted to 25.5°C after hatching. Alternatively, L1s were directly picked from 15 to 25.5°C. For the analysis of spermatocyte meiosis defects, him-8 doubles of mat mutants were grown at 16°C until the late L3 larval stage. Males were then picked to fresh plates and grown at 25.5°C until they reached early adulthood. For the analysis of embryos and gonads within somatically unaffected adults, young adult hermaphrodites with few, if any, embryos in their uteri were upshifted to 25.5°C for 6–7 h. Embryos and gonads were then isolated for analysis, as described below. For many alleles, it proved essential to maintain the incubators at 25.5 ± 0.5°C to attain fully penetrate mutant phenotypes.
Immunohistochemistry and Other Phenotypic Analysis
For the analysis of early embryos and hermaphrodite gonads, upshifted adult hermaphrodites were transferred to a 5-μl drop of egg buffer (Edgar 1995) on a slide subbed with poly-l-lysine (Sigma-Aldrich). Embryos and gonads were released using 27.5 gauge needles, freeze–cracked, and processed for antibody staining (Miller and Shakes 1995). For tubulin immunostaining, embryos were fixed in −20°C methanol for at least 1 h. After three washes in PBS, the slides were blocked for 1 h in PBS containing 0.5% BSA and 0.1% Tween 20 before a 2 h room temperature incubation with 1:100 FITC-conjugated anti–α-tubulin antibodies (DM1A; Sigma-Aldrich) (Blose et al. 1984). 4,6-diamidino-2-phenylindole (DAPI) (1 μg/ml) was added during the last 5 min. After a brief wash in PBS, the slides were mounted using either Anti-Fade (Molecular Probes) or Gel/Mount (Biomeda Corp.). Other immunostaining protocols were similar. Specimens were incubated at room temperature with 1:100 anti-phosphohistone H3 polyclonal antibody (Upstate Biotechnology) (Hendzel et al. 1997) for 3 h and rhodamine-conjugated anti–rabbit secondary IgG (Sigma-Aldrich) for 2 h. Specimens were incubated with 1:500 MPM-2 monoclonal antibody (Dako) (Davis et al. 1983) for 16–20 h at 4°C and with rhodamine-conjugated anti–mouse IgG (Jackson ImmunoResearch Laboratories) for 2 h at room temperature. In some cases, DAPI was added directly to the mounting media.
Meiotic sperm spreads were generated for Hoechst/DIC analysis by dissecting male gonads in egg buffer (Edgar 1995) containing 100 μg/ml of lipid soluble DNA-binding Hoechst dye 33342 (Sigma-Aldrich). A coverslip was placed over the isolated gonad and gentle pressure was applied to generate a monolayer of spermatocytes and spermatids. The spreads were immediately analyzed using Nomarski/DIC optics while simultaneously illuminating the specimen with UV epifluoresence to visualize the DNA. For DAPI/tubulin immunostaining, sperm spreads were generated on ColorFrost Plus slides (Fisher Scientific) in buffer without Hoechst. The samples were freeze–cracked in liquid nitrogen before a 12–20-h incubation in −20°C methanol. Tubulin immunostaining was carried out as above.
Additional somatic and germline defects of L1 upshifted animals were analyzed either under DIC optic in living, agar-mounted adult animals (Sulston and Horvitz 1977), or by UV epifluorescence in whole-mount, DAPI-stained animals that had been fixed in Carnoy II fixative (6:3:1 ethanol/acetic acid/chloroform).
Physical Mapping of emb-27
emb-27 lies within the gene subcluster of LGII (Cassada et al. 1981). Fine-scale genetic mapping studies using a large set of overlapping deficiencies placed emb-27 to the right of unc-105 (Sigurdson et al. 1984) and to the left of mig-5 (Guo 1995). The cloning and physical mapping of unc-105 and mig-5 to the cosmids C41C4 (Liu et al. 1996) and TO5C12 (Guo 1995), respectively, positioned emb-27 to a region of three overlapping cosmids, C41C4, F10B5, and TO5C12. Information from the C. elegans Sequencing Center was used to identify candidate genes from this region.
RNA-mediated Interference (RNAi)
An expressed sequence tag clone of F10B5.6 (CEESZ14) was used as the template for CeCdc16 cDNA synthesis. CEESZ14 is a truncated cDNA that contains the last 20% of the predicted F10B5.6 open reading frame and the 3′ untranslated region. M13 forward and reverse primers were used to amplify this cDNA insert. Sense and antisense RNAs were synthesized using a T3 and T7 in vitro transcription kit (Ambion). The RNA synthesis reactions were mixed and incubated at 70°C to denature the RNAs and then slow cooled at room temperature to anneal the complementary strands. Young wild-type adult hermaphrodites were microinjected by standard protocols (Mello et al. 1991) with dsRNA, and they were allowed to recover 12 h before analysis by DIC microscopy or fixation for DNA staining. Similar methods were used to analyze the RNAi phenotypes of other emb-27 gene candidates.
cDNA Synthesis and DNA Sequencing
Total RNA was prepared from wild-type and emb-27(g48) hermaphrodites by standard RNA isolation methods. First strand cDNA was prepared with a reverse transcription–PCR kit according to the manufacturer's instructions (Gene Amp Reverse Transcription-PCR; PerkinElmer). F10B5.6-specific cDNAs were PCR amplified with oligonucleotide primers corresponding to either the SL1 trans-spliced leader RNA or the predicted start codon coupled with a primer that contained the predicted translation stop codon. The PCR products were gel purified using Wizard columns (Promega) and cloned into a pBluescript vector (Stratagene). Automated DNA sequencing of the F10B5.6 cDNA was performed using standard methods. A contiguous consensus contig was generated by the CAP Sequence Assembly program (Huang 1992) accessed via the Baylor College of Medicine Search Launcher. Two independent PCR reactions were sequenced for both wild-type and g48, and they were used to determine the consensus sequence of both the wild-type and g48 F10B5.6 gene sequence.
emb-27(g48) Mothers Produce Meiotic One-Cell Arrested Embryos
Our initial interest in mutants that arrest at the metaphase to anaphase transition of meiosis I arose from our analysis of the maternal effect embryonic lethal (Mel) mutant emb-27(g48). The emb-27(g48) mutant was originally isolated in a genetic screen for ts Mel mutants (Cassada et al. 1981). emb-27(g48) was reported to produce embryos that arrested at the one-cell stage and failed to form polar bodies, the discarded karyocytes of oocyte meiotic divisions (Denich et al. 1984). Although the terminal phenotype of the mutant emb-27(g48) embryos has been reported to be somewhat variable, growth under strict temperature conditions (see Materials and Methods) resulted in a highly penetrant and consistent meiotic one-cell arrest.
Whereas wild-type hermaphrodites contain rapidly cleaving embryos of many different ages, emb-27(g48) mutant hermaphrodites contain only one-cell embryos (Fig. 1A and Fig. B). Typically, wild-type hermaphrodites have only a single newly fertilized embryo that is at the same meiotic one-cell stage as the entire clutch of emb-27 embryos. A similar early arrest phenotype has been observed for embryos in which the expression of three C. elegans cdc-25 genes has been disrupted by RNAi; both emb-27(g48) and cdc-25 (RNAi) embryos arrest at the one-cell stage without forming pronuclei (Ashcroft, N., and A. Golden, manuscript in preparation). This phenotype is distinct from cleavage-defective mutants such as air-2 and cyk-1, which arrest at the one-cell stage but nevertheless complete meiosis, exit M phase, and produce pronuclei (Schumacher et al. 1998; Swan et al. 1998).
Before fertilization, emb-27 oocytes are indistinguishable from their wild-type counterparts. When stained with the DNA dye DAPI, diakinetic emb-27 oocytes were found to contain a normal complement of six bivalents, indicating that the early meiotic events of homologue pairing, homologous recombination, and chromosome condensation occurred normally. As in wild-type animals, mature emb-27 oocytes undergo nuclear envelope breakdown just before ovulation. After fertilization, they exhibit many, but not all, of the normal events of egg activation. For instance, the fertilized emb-27 (g48) oocytes assume an oval-shaped morphology and initiate eggshell deposition (Fig. 1 B). However, none of these mutant embryos progress beyond the normal meiotic one-cell stage (n > 200) (Fig. 1B and Fig. D). DAPI staining of the mutant g48 embryos revealed that both the sperm chromosomes and oocyte chromosomes remain condensed and positioned near the cortex. This persistent pattern, a cluster of tightly arrayed oocyte chromosomes and a single sperm chromatin mass (Fig. 1 D), is identical to what occurs during the normally transient, meiotic one-cell stage of wild-type embryogenesis (Fig. 1 C). Likewise, the eggshells of emb-27 embryos remain osmotically sensitive, another characteristic of wild-type meiotic one-cell embryos. These features distinguish emb-27 embryos from unfertilized oocytes, which remain spherical in shape, fail to form eggshells, and contain a single, centrally located mass of endoreduplicating oocyte chromosomes.
emb-27 Encodes a Subunit of the APC/C
Genetic and physical map data positioned emb-27 within a three cosmid region on chromosome II (Materials and Methods). Because our attempts to identify emb-27 by germline transformation rescue proved inconclusive, information from the C. elegans Sequencing Center was used to identify emb-27 candidate genes. Of these genes, only F10B5.6 yielded an emb-27–like, one-cell arrest phenotype (Fig. 1 E) by RNAi. This result prompted us to sequence F10B5.6 from the emb-27(g48) mutant.
Reverse transcription–PCR was used to amplify the full-length cDNA of F10B5.6 from wild-type and emb-27 (g48) animals. Sequencing and translational analysis of the wild-type cDNA confirmed the Genefinder predicted structure of F10B5.6 as a 655 amino acid protein, and analysis of the g48 cDNA identified a C to T base change at position 1,150 of the cDNA sequence. This change results in a His354Tyr alteration in the amino acid sequence (Fig. 1 G). A BLAST search (Altschul et al. 1990) using the entire F10B5.6 sequence identified the CDC16 gene from Saccharomyces cerevisiae as the closest match (20% identity and 40% similarity over 561 amino acids).
CDC16 is an evolutionarily conserved component of the APC/C (King et al. 1995; Morgan 1999). Structurally, the Cdc16p subunit contains 10 tetratricopeptide repeats (TPR) (Sikorski et al. 1990), tandem arrays of which form an amphipathic groove that mediates protein–protein interactions (Blatch and Lassle 1999). TPR proteins are typically described by the number and distribution of their TPR repeats, and F10B5.6 contains the same pattern of TPR repeats as Cdc16p (S. cerevisiae) and its Saccharomyces pombe ortholog, Cut9p (Fig. 1 F). The g48 mutation lies within TPR6, the TPR within Cdc16p that exhibits the highest degree of interspecies sequence identity (Samejima and Yanagida 1994; this study) (Fig. 1 G). The C. elegans sequence is 29% identical and 65% similar to the S. cerevisiae sequence in this region. The mutated His354 is conserved in all Cdc16 proteins sequenced to date, but it is not one of the conserved hydrophobic residues that defines the TPR motif itself. Instead, this histidine is positioned on the hydrophilic face of α helix A (Das et al. 1998), and, thus, the mutation is predicted to alter both the hydrophobicity and charge of this helical face. Taken together, these data indicate that emb-27 encodes the C. elegans CDC16 ortholog, and that this APC/C subunit is essential for proper APC/C function.
Identification and Isolation of Additional emb-27–like Mutants
Because Cdc16 is only 1 of the 8–12 APC/C subunits, it seemed likely that genetic screens for similar one-cell arrest mutants would identify mutants in additional APC/C and APC/C-related genes. Using the emb-27(g48) phenotype as a guide, we screened preexisting collections of ts Mel mutants for additional members of this class. From several different mutant collections (Miwa et al. 1980; Wood et al. 1980; Cassada et al. 1981), we identified two such genes: emb-1(hc57, hc62) and emb-30(g53) (Fig. 2). Although emb-1 has not yet been cloned, emb-30 encodes an ortholog of APC4 (Furuta et al. 2000).
We also initiated a large scale screen for new one-cell arrested ts mutants. Our goal was to isolate additional alleles of emb-1, emb-27, and emb-30, as well as novel members of this phenotypic class. To facilitate the isolation of Mel mutants, screens were carried out in a genetic background of an egg laying–defective mutant (lin-2) (Kemphues et al. 1988). The mutant phenotypes were scored in hermaphrodites that had been upshifted to the restrictive temperature of 25°C as L4 larvae. Of ∼1.9 million haploid genomes screened, >1,400 ts Mel mutants were isolated. Of these, 44 mutants produced one-cell embryos when homozygous L4s were shifted to 25°C, and 32 of these exhibited meiotic progression defects similar to those of emb-27(g48). Mapping crosses and complementation tests revealed that seven of these mutants are new alleles of emb-27 (6 alleles) and emb-30 (1 allele), whereas the remaining 25 alleles define three new complementation groups: mat-1 (6 alleles), mat-2 (7 alleles), and mat-3 (12 alleles). The genetic map positions of these genes are shown in Fig. 2.
Meiotic One-Cell Mutant Embryos Are Blocked in Metaphase of Meiosis I
Between fertilization and the first mitotic division, the one-cell stage of C. elegans embryogenesis encompasses the meiotic divisions of the oocyte, a round of DNA synthesis, the conjunction of the pronuclei, and anterior–posterior polarization of the embryo (Albertson 1984; Albertson and Thomson 1993; Kemphues and Strome 1997; Sadler and Shakes 2000). Fig. 3 shows DNA and tubulin images of the events that lead up to the first mitosis in wild-type embryos. Before fertilization, the primary oocytes undergo a prolonged pause in diakinesis of meiosis I. At this stage, they contain six highly condensed bivalents within a nuclear envelope and a uniform meshwork of cytoplasmic tubulin (Fig. 3A and Fig. B) (Albertson 1984). Immediately before fertilization, they undergo meiotic maturation, which includes nuclear envelope breakdown (McCarter et al. 1999). After fertilization, the oocyte-derived bivalents align and congress to form a metaphase plate with the five autosomal bivalents surrounding the sex chromosome bivalents in an axial orientation. This arrangement appears as either a “pentagonal array,” or, in side view, as a cluster of two or three axially aligned bivalents (Fig. 3 C). The associated meiotic spindle is a barrel-shaped structure that lies adjacent and parallel to the cortex (Fig. 3 D) (Albertson and Thomson 1993). During this time, the quiescent sperm chromatin remains as a single mass of condensed DNA near its site of entry (Sadler and Shakes 2000). During anaphase, the spindle rotates to its telophase position, perpendicular to the cortex (Fig. 3E and Fig. F), before extruding one set of oocyte chromosomes into the polar body (Fig. 3 G). Meiosis II proceeds in a similar manner (Fig. 3G and Fig. H) (Albertson 1984). After these meiotic divisions, the maternal and paternal chromosomes decondense, are encased within nuclear envelopes, and undergo DNA synthesis (Sadler and Shakes 2000). During this same period, the sperm centrioles duplicate and begin nucleating microtubules (Fig. 3 J) (Albertson 1984). Subsequently, the zygote enters a period of transitional prophase during which the maternal pronucleus migrates posteriorly to meet the paternal pronucleus (Fig. 3I and Fig. J). The joined pronuclei then move centrally before initiating mitosis (Fig. 3 L).
To further investigate the mutant defects, oocytes and embryos of adult-upshift mutant mothers were examined. No prefertilization defects were detected; DAPI staining revealed the expected six bivalents per oocyte and maturation defects were undetectable by DIC optics. To analyze defects after fertilization, wild-type and mutant embryos were costained with DAPI and anti-tubulin antibodies. Embryos within the uteri of wild-type mothers ranged in age from the 1–50-cell stage (Fig. 4A and Fig. B), and, because the 1-cell stage lasts <1 h at 25°C (Ward and Carrel 1979; McCarter et al. 1999), only one or two of these embryos were at the one-cell stage of development. In contrast, each of the mutant mothers were filled with 1-cell embryos, all of which were arrested at the same stage of development (Fig. 4, C–N, compare with Fig. 3C and Fig. D). Within the mutant embryos, the oocyte chromosomes had properly congressed and aligned on an acentriolar, barrel-shaped meiotic spindle. However, no evidence of a metaphase to anaphase transition was observed: the homologues remained attached, and the spindle failed to rotate from its initial position parallel to the cortex. On the other side of the embryo, the sperm chromatin mass remained hypercondensed, and the sperm centrosomes remained quiescent. As assessed by DAPI/tubulin staining, all 32 alleles exhibited an identical oocyte meiosis defect; the embryos arrest with what appears to be a normal meiosis I metaphase spindle (compare Fig. 3C and Fig. D, with Fig. 4, C–N). Because these meiotic one-cell mutants share a common metaphase-arrest defect, we call them “mat” for metaphase to anaphase transition defective.
mat Mutant Embryos Remain in a Prolonged M phase–like State
Because cell cycle dynamics are ultimately regulated by the activation and inactivation of cyclin-dependent kinases (CDKs) (Gitig and Koff 2000), the phosphorylation state of direct and indirect CDK targets can also be used to determine the stage of cell cycle arrest. For instance, histone H3 is phosphorylated during M phase entry and subsequently dephosphorylated before M phase exit (Su et al. 1998; Wei et al. 1998, Wei et al. 1999). Therefore, to test whether the mutant embryos remain in a prolonged M phase state, embryos were analyzed using an antibody against phosphorylated histone H3 (phospho-H3). In both wild-type and mutant embryos, anti–phospho-H3 antibodies intensely stained oocyte chromosomes in their metaphase configuration (Fig. 5). In the mutants, the oocyte chromosomes continued to stain even in the oldest in utero embryos, suggesting that the mutant embryos enter, but never leave, M phase. Note that this antibody does not recognize the highly condensed sperm chromatin mass in either wild-type or mutant embryos (Fig. 5).
Mutant embryos were also analyzed using the MPM-2 monoclonal antibody that recognizes various mitotic phosphoproteins (Davis et al. 1983). The MPM-2 epitope is widely conserved, as this antibody stains kinetochores, centrosomes, and microtubules during early, but not late, M phase (Vandre et al. 1984; Hecht et al. 1987; Vandre and Borisy 1989; Hirano and Mitchison 1991; Renzi et al. 1997). As shown in Fig. 6, MPM-2 stains a “halo” of material around or, in some case, between the congressed oocyte meiotic chromosomes in both wild-type (Kitagawa and Rose 1999) and mutant embryos. MPM-2 also stains the sperm chromatin mass (Fig. 6). In the mutants, MPM-2 continued to stain the chromosomes in older embryos, a further indication that these aging embryos remain blocked in M phase.
Under stringent conditions, every allele from this ts mutant collection exhibits a block in both the meiosis I metaphase to anaphase transition and in M phase exit. Nevertheless, after 3–4 h at 25.5°C, the oocyte and sperm DNA within the aging mutant embryos leave their position near the cortex and move centrally, and the metaphase chromosomes begin to unravel. Importantly, this DNA unraveling is distinct from DNA decondensation, since the unraveled DNA continues to stain with the phospho-H3 antibody (Fig. 5 F, bottom embryo), and it never become encased within a nuclear envelope (data not shown).
When mutant mothers were grown under suboptimal conditions (e.g., growth at 20–24°C or after brief upshifts), their mutant embryos were sometimes observed to exit M phase and reform nuclear envelopes in the absence of chromosome separation. Other embryos even underwent a few rounds of mitotic cell divisions. The particular conditions required to produce these hypomorphic phenotypes are allele specific, and the hypomorphic defects themselves are consistent with the previously reported phenotypes of both emb-27(g48) and emb-30(g53) (Cassada et al. 1981), as well as emb-1(hc62 and hc57) (Miwa et al. 1980).
Mutant Spermatocytes Are Also Blocked in Metaphase of Meiosis I
In C. elegans, the first meiotic division of the oocyte takes place on an acentriolar barrel-shaped meiotic spindle and culminates in a highly unequal division in which one set of homologues are discarded into a tiny polar body. In contrast, the first meiotic division of a primary spermatocyte takes place on a centriole-based, mitotic-like spindle, and, though cytokinesis is often incomplete, the division culminates in the symmetric division of the primary spermatocyte into two secondary spermatocytes (Ward et al. 1981) (Fig. 7 A). Unlike oocytes, in which the first meiotic division only occurs after fertilization, spermatocytes initiate their first meiotic division autonomously as soon as they mature and separate from the syncytial gonad. This first meiotic division is followed by a round of centriole duplication and the assembly of a bipolar meiosis II spindle, orthogonal to the first division axis. The separation of sister chromatids during meiosis II is accompanied by an unusual asymmetric division (spermatid budding) during which the two haploid sperm bud from a large residual cytoplast (Fig. 7 A).
Given these striking differences between oocyte and spermatocyte meiosis, we investigated whether the mat genes functioned in both processes. Testes from young adult mutant males, which had been upshifted to 25.5°C as L3 larvae, were isolated and processed for DIC/Hoechst analysis. Such “sperm spreads” from wild-type males contain cells in all stages of spermatogenesis, including primary spermatocytes, secondary spermatocytes, budding figures, and haploid sperm (Fig. 7 B). In contrast, mat mutant spreads exhibit a striking abundance of primary spermatocytes and sperm (Fig. 7 C). Notably, secondary spermatocytes are completely absent, and the DNA staining patterns suggest that meiosis in the mutant primary spermatocytes fails to progress past meiosis I. Despite this apparent meiosis I arrest, cells that resemble haploid sperm still form, but they lack DNA (which we refer to as anucleate). Similar sperm spreads were seen for most mat alleles (data not shown) under L3 shift-up conditions, with the striking exceptions that mat-3 (or172, or180, and or187) and emb-1 (hc62 and hc57) males produce morphologically normal secondary spermatocytes and haploid sperm and can sire viable offspring when mated to feminized hermaphrodites (data not shown) (Sadler, P., D. Fox, A. Pletcher, and D. Shakes, unpublished observations). As previously reported for emb-27 and emb-30 males (Sadler and Shakes 2000), affected mat-1, mat-2, and mat-3 males are unable to sire viable offspring, however, their anucleate sperm are surprisingly functional in that they can activate, crawl, and even fertilize oocytes.
To further understand the meiotic defects, we analyzed stage-specific DNA and tubulin structures in wild-type and mutant sperm spreads. In wild-type animals (Fig. 7D and Fig. E), sperm spreads reveal a diversity of microtubule structures ranging from the large, closely opposed asters of the metaphase primary spermatocytes to the highly dynamic spindles of the secondary spermatocytes and budding figures. In mat mutants, including mat-2(ax143) (Fig. 7F and Fig. G), sperm spreads are dominated by large spermatocytes, each of which contains a metaphase array of chromosomes and a bipolar, astral, meiosis I metaphase spindle. In enlarged views (Fig. 7, H–K), these spindles are closely associated with properly congressed and aligned metaphase chromosomes. Some of the mutant spindles eventually elongate, but homologue separation and other anaphase events never occur. Thus, with the possible exception of emb-1, whose two alleles lack spermatocyte defects, these data indicate that every mat gene is required for the meiosis I metaphase to anaphase transition in both oocytes and spermatocytes.
The mat Genes Are Also Required for Germline Mitosis
Our analysis of oocyte and spermatocyte meiosis in the mutants suggested that the mat gene products function in a shared step of these two structurally dissimilar meiotic processes. To address whether these mat genes function specifically to separate axially aligned homologues during meiosis I or whether they also function to separate sister chromatids during mitosis, we examined the mitotic divisions of germline nuclei within adults. Because germline mitotic divisions continue at a time when the animal's somatic cells have ceased to divide (Beanan and Strome 1992), they can be studied in adult upshifted mutants in the absence of potential complications from somatic defects. To test for mitotic defects, young adult hermaphrodites were upshifted to 25°C for 6 h. When isolated wild-type gonads were immunostained with phosphohistone H3 antibodies, they were found to contain one to six mitotic germ cell nuclei in the distal region of each gonadal arm (Fig. 8, A–D, Table). In an allele-specific manner, mutant gonads had either slightly or greatly elevated numbers of phospho-H3 staining mitotic germ cell nuclei (Fig. 8; Table). DAPI staining of these mitotic nuclei revealed that most of the phospho-H3–positive nuclei are in metaphase (Fig. 8, E–G), though anaphase figures are also common, particularly in weaker alleles. We believe that the excess in mitotic figures stems from an extended block or pause in metaphase, rather than an increased cell cycle rate, since upshifts of longer duration result in reduced rather than tumorous germlines (data not shown).
Although these initial studies indicated a role for mat genes in the “maintenance” of the mitotic germline pool in adults, they did not address whether the mat genes are also required for the initial proliferation of the germline. Newly hatched C. elegans larvae contain only two germline progenitor cells, Z2 and Z3, that must proliferate during larval development to generate the >1,000 germ cell nuclei of the mature adult gonad (Kimble and White 1981). To test whether mat genes are required for the initial expansion of the germline, mutants were upshifted as newly hatched L1 larvae. For some alleles, the earlier upshift conditions did not significantly alter the mutant phenotype; these Mel adults had fully proliferated germlines and produced large broods of one-cell arrested embryos (Table). For other alleles, these same conditions caused moderate to severe defects in germline proliferation (Table and data not shown). In the most severe cases, L1 upshifts resulted in sterile (Ste) adults with fewer than 50 germ cells and no apparent gametes. Similar defects in mitotic germline proliferation have been reported for null alleles of emb-30 (Furuta et al. 2000).
In Embryos and Larvae, the mat Genes Are Also Essential for Somatic Cell Divisions
To investigate whether the mat genes are also required for mitotic cell divisions of the soma, mutant hermaphrodites and males were upshifted as L1 larvae and examined for potential somatic defects. Although our analysis did not include all 32 alleles, we observed that a subset of alleles within the mat-1, mat-2, mat-3, and emb-27 complementation groups exhibit defects in somatic development. For instance, for mat-1(ax144) animals, >40% of the hermaphrodites have protruding vulvae, and >90% of males have tails with missing or abnormal ray structures (data not shown). Similar somatic defects were reported for a subset of emb-30 alleles (Furuta et al. 2000). For three mat-3 alleles, the animals are also uncoordinated, suggesting defects in the proliferation of cells that make up the ventral nerve cord. The hermaphrodite vulva, male tail, and ventral nerve cord are all structures that require substantial numbers of larval cell divisions, and the spectrum of observed phenotypes resemble those of cell cycle mutants in which postembryonic divisions have been disrupted (O'Connell et al. 1998; Boxem et al. 1999; Park and Krause 1999; Woollard and Hodgkin 1999). We have not yet determined whether these somatic defects are caused by arrest or delays in the mitotic cell cycle.
In an attempt to generate stronger somatic defects, two-cell embryos, which had completed both oocyte meiosis and the first mitotic division at 16°C, were dissected from their mothers and directly upshifted to 25°C. Our complete set of ts alleles were tested in this manner, and, in most cases, these two-cell embryos underwent normal embryogenesis and developed into adults. Depending on the allele, the adults either produced broods of one-cell embryos (Mel) or were Ste (Table). In most cases, the phenotype of the two-cell and the L1 larval upshifts did not differ; in others, embryonic upshifts enhanced the germline proliferation, hermaphrodite vulva, male tail, and/or uncoordinated defects described above (data not shown). The unexpected mildness of the observed somatic defects, particularly in upshifted two-cell embryos, may suggest that these mat genes function in only a subset of mitotic divisions. Alternatively, our results may be explained by maternal rescue, gene redundancy, or the fact that these ts alleles are unlikely to be genetic nulls.
In summary, we have isolated and characterized a large set of ts mutant alleles that define five complementation groups and share a common defect: mutant mothers shifted to the nonpermissive temperature of 25°C produce embryos that arrest at the meiotic one-cell stage. Our premise, that these affected one-cell embryos reach a normal meiosis I metaphase state, but then fail to progress to anaphase, is supported by the following observations: (a) the oocytes undergo normal maturation events and, after fertilization, their chromosomes align and congress in a metaphase pentagonal array on a morphologically normal meiotic spindle, (b) in such embryos, the sperm chromatin remains condensed and the sperm microtubule organizing center remains quiescent, and (c) neither meiotic anaphase figures nor polar bodies are ever observed. In addition, the stable presence of phosphohistone H3 and MPM-2 epitopes indicates that the mutant embryos are permanently arrested in an M phase–like state. These results suggest that functional meiotic CDKs drive the mutant embryos into metaphase of meiosis I, but because these CDKs are not subsequently deactivated, the mutant embryos are unable to transition out of a metaphase state. Thus, we have chosen the name “mat” (defined above) as an appropriate designation for this general mutant class.
Importantly, the metaphase to anaphase transition defects in these mutants are not restricted to oocyte meiosis I. Analogous defects are observed during spermatogenesis: primary spermatocytes undergo normal budding from the gonadal syncytium, undergo nuclear envelope breakdown, but then arrest in a normal meiosis I metaphase state and never form secondary spermatocytes. Thus, the same fundamental defect is seen in both spermatocyte and oocyte meiosis, despite the fact that spermatocytes employ astral, rather than anastral spindles. On the other hand, the developmental consequences of this common meiosis I block are strikingly dissimilar. The mutant oocytes appear to be blocked from further development as their sperm chromatin remains condensed and their sperm-contributed microtubule organizing centers remain quiescent. In contrast, differentiation of the mutant spermatocytes proceeds with the eventual formation of motile, albeit anucleate, spermatozoa.
These mutants also display metaphase to anaphase transition defects during mitosis. Most dramatically affected are the mitotic divisions of proliferating germ cells; upshifted adults accumulate an excessive number of phospho-H3–staining germ cell nuclei, and upshifted larvae exhibit moderate to severe defects in germ cell proliferation. However, unlike the complete metaphase blocks observed during meiosis, the metaphase block during germ cell mitosis is frequently incomplete and results in an M phase delay. The identification of defects in somatic tissues, such as the male tail and the hermaphrodite vulva, suggests that the mat genes are also required for mitotic divisions of the soma.
Each of the mat genes, except for emb-1, is represented by an allelic series that encompasses the full range of described metaphase to anaphase transition defects. Because identical mutant defects are observed in multiple alleles of multiple genes, they are likely to reflect important functions of this gene class. Thus, our data implies that a common mechanism governs the transition from metaphase to anaphase, regardless of whether this transition (a) involves the separation of homologues or sister chromatids, (b) is mediated by anastral or astral spindles, (c) occurs within syncytial or nonsyncytial cells, or (d) involves germline or somatic cells. On the other hand, whereas all of the mutant alleles have defects in oocyte meiosis and most have defects in spermatocyte meiosis, many fewer exhibit defects in the mitotic divisions of either the germline or soma. Furthermore, we observed strong metaphase blocks during meiosis I, but primarily M phase delays during germline mitosis. Thus, whereas the mat genes are required for all of these various cell divisions, meiotic divisions may be particularly sensitive to low levels of gene product. Alternatively, the design of our mutant screens may have favored the isolation of mutants with lesions in meiotic-specific or germline-specific domains. Sequence analysis of the mutant lesions may preferentially support one of these two explanations, and, in the latter case, identify specific functional subdomains within each protein.
What is the molecular nature of these genes that are required for the metaphase to anaphase transition during various types of cell divisions? Evidence from the cell cycle literature suggests that mitotic metaphase blocks can stem either from defects in the machinery that drives the metaphase to anaphase transition or from spindle and/or kinetochore attachment defects that secondarily trigger the metaphase checkpoint system. Recent studies suggest that this checkpoint is functioning within one-cell C. elegans embryos (Kitagawa and Rose 1999), but, because the mat mutants assemble morphologically normal metaphase plates and spindles, we suspect the mat genes encode components of the cell cycle machinery per se. Consistent with this hypothesis, we report that emb-27 encodes an ortholog of the APC/C subunit, Cdc16. Molecular analysis of the other mat genes is ongoing, but we have recently discovered that mat-1 encodes an ortholog of another APC/C gene, cdc27 (Schumacher, J., D. Shakes, and A. Golden, manuscript in preparation), and Furuta et al. 2000 report that emb-30 encodes APC4. Thus, the Mat phenotype appears to be an excellent predictor of APC/C and APC/C-related genes, and it is not surprising that emb-1, mat-2, and mat-3 all map to regions in which APC/C subunits have been identified.
It is somewhat surprising that this large mutant screen, which is at or near saturation, yielded multiple alleles of only five genes (emb-1 was not reisolated), all of which are either known or likely to encode conserved APC/C components. First, this screen was expected to identify several meiosis-specific APC “accessory proteins” or regulators that enable the APC/C to separate homologues during meiosis I without disrupting the centromeric sister chromatid cohesions that are APC/C's primary, but indirect target, during mitosis and meiosis II. Although our results do not rule out the existence of such modifiers, they do indicate that such modifiers do not easily mutate to temperature sensitivity. This result suggests that APC/C functions largely in the same capacity in distinct types of cell divisions. Second, even if all six mat genes prove to encode APC/C subunits, there are at least nine APC/C subunits encoded within the C. elegans genome (King et al. 1995, Yu et al. 1998; Zachariae et al. 1998; Furuta et al. 2000; Wille, L., and D. Shakes, manuscript in preparation). It is not clear why additional genes were not hit, but similar ts screens in yeast likewise identified only a subset of APC/C genes. Thus, something about either the function or molecular structure of the individual subunits must make them more or less likely to be isolated in such ts screens. This possibility is supported by studies in S. cerevisiae in which only a small number of essential genes could be mutated to be ts (Kaback et al. 1984; Harris and Pringle 1991).
The phenotypic analysis of this large set of ts mat mutants and the molecular identification of emb-27 as an ortholog of Cdc16 have enabled us to define an essential role for APC/C in a variety of specialized cell divisions, including the first meiotic division of both oocytes and spermatocytes. Importantly, our analysis reveals that all of these metaphase to anaphase transitions are driven by a common cellular mechanism, and that many or all of the individual APC/C subunits are essential for M phase progression in these various cell types. Ts mutants proved critical to our analysis, since meiotic functions could not have been analyzed in nonconditional mutants with defects in germline proliferation. In continuing studies, analysis of the molecular lesions associated with each allele should reveal which protein domains are important for general and/or cell-type–specific functions. Additionally, screens for genetic suppressers may identify additional components that act in each of these pathways.
Many thanks to Michael Krause, Ann Corsi, Orna Cohen-Fix, and members of the Bowerman, Golden, Seydoux, and Shakes laboratories for helpful discussions and comments on the manuscript. We also gratefully acknowledge Neville Ashcroft, Mary Kosinski, Ilsa Kaattari for their excellent technical support, A.R. Kerlavage (The Institute for Genomic Research) for providing the CEESZ14 cDNA, and the C. elegans Genetics Center (University of Minnesota), funded by the NIH National Center for Research Resources, for providing mapping strains.
This work was supported in part by grants from the National Science Foundation (IBN92653092 to D. Shakes), the National Institutes of Health (IR15GM60359-01 to D. Shakes, 1RO1GM58017-02 to B. Bowerman), the American Cancer Society (to G. Seydoux, PF-4444 to D. Hamill), the Jeffress Memorial Trust (J387 to D. Shakes), the Packard Foundation (to G. Seydoux), the March of Dimes (to G. Seydoux), and Howard Hughes Medical Institute (Undergraduate Biological Sciences Education grant to the College of William and Mary).
A. Golden and P.L. Sadler contributed equally to this work.
Abbreviations used in this paper: APC/C, anaphase promoting complex or cyclosome; CDK, cyclin-dependent kinase; DAPI, 4,6-diamidino-2-phenylindole; DIC, differential interference contrast; Mel, maternal effect embryonic lethal; RNAi, RNA-mediated interference; Ste, sterile; TPR, tetratricopeptide repeat; ts, temperature sensitive.