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Genetics. 2009 Mar; 181(3): 875–887.
doi: 10.1534/genetics.108.097741
PMCID: PMC2651061
PMID: 19104074

Mutations in the Chromosomal Passenger Complex and the Condensin Complex Differentially Affect Synaptonemal Complex Disassembly and Metaphase I Configuration in Drosophila Female Meiosis

Tamar D. Resnick,*,1 Kimberley J. Dej,*,2 Youbin Xiang,† R. Scott Hawley,†‡ Caroline Ahn,*,3 and Terry L. Orr-Weaver*,4
Author information Article notes Copyright and License information PMC Disclaimer

Abstract

Production of haploid gametes relies on the specially regulated meiotic cell cycle. Analyses of the role of essential mitotic regulators in meiosis have been hampered by a shortage of appropriate alleles in metazoans. We characterized female-sterile alleles of the condensin complex component dcap-g and used them to define roles for condensin in Drosophila female meiosis. In mitosis, the condensin complex is required for sister-chromatid resolution and contributes to chromosome condensation. In meiosis, we demonstrate a role for dcap-g in disassembly of the synaptonemal complex and for proper retention of the chromosomes in a metaphase I-arrested state. The chromosomal passenger complex also is known to have mitotic roles in chromosome condensation and is required in some systems for localization of the condensin complex. We used the QA26 allele of passenger component incenp to investigate the role of the passenger complex in oocyte meiosis. Strikingly, in incenpQA26 mutants maintenance of the synaptonemal complex is disrupted. In contrast to the dcap-g mutants, the incenp mutation leads to a failure of paired homologous chromosomes to biorient, such that bivalents frequently orient toward only one pole in prometaphase and metaphase I. We show that incenp interacts genetically with ord, suggesting an important functional relationship between them in meiotic chromosome dynamics. The dcap-g and incenp mutations cause maternal effect lethality, with embryos from mutant mothers arrested in the initial mitotic divisions.

ORGANISMS that undergo sexual reproduction utilize a specialized cell cycle, meiosis, to generate haploid gametes. Precise partitioning of the genome in meiosis is essential so that diploidy is reestablished upon fertilization, which is critical for embryonic development (Hassold and Hunt 2001). Meiosis employs distinct regulatory mechanisms such that the DNA is replicated exactly once and then divided twice without an additional intervening round of replication.

In preparation for meiotic divisons, homologs pair and, in many systems, a proteinaceous structure, the synaptonemal complex (SC), forms an axis between homologs and regulates meiotic recombination (Page and Hawley 2003). Crossover events generate covalent linkages between homologs. These, in combination with sister-chromatid cohesion distal to the chiasmata (the physical structures resulting from crossing over), allow homologs to remain physically attached after SC disassembly and to thereby coordinate their movements.

In meiosis I, homologs biorient on the spindle while sister chromatids coorient toward a single pole (reviewed in Petronczki et al. 2003). Release of cohesion distal to the chiasmata at the onset of anaphase I allows homologs to move apart; maintenance of centromere cohesion holds sister chromatids together as they travel toward a single spindle pole. The enduring attachment between sister chromatids is essential for them to biorient on the spindle in meiosis II. Centromere cohesion is severed at the onset of anaphase II and sister chromatids segregate, in a manner more similar to mitosis.

Progression through the meiotic program relies critically on activity of both meiosis-specific factors and proteins that are also essential in mitosis. Study of the meiotic roles of proteins required in mitosis has been hampered by a shortage of alleles weak enough to allow development of an animal, but strong enough to reveal meiotic phenotypes. The condensin complex and the chromosomal passenger complex are important regulators of chromosome dynamics in mitosis, but their roles in meiosis remain much less characterized (Vagnarelli and Earnshaw 2004; Hirano 2005).

The condensin complex is required in mitosis for resolution of sister chromatids, and its absence results in fuzzy chromosome morphology and lagging chromatin in anaphase (Strunnikov et al. 1995; Freeman et al. 2000; Lavoie et al. 2000; Ouspenski et al. 2000; Steffensen et al. 2001; Hagstrom et al. 2002; Hagstrom and Meyer 2003; Dej et al. 2004). Condensins also play roles in condensation, though they are not essential for tight compaction of chromosomes in metazoans. Five conserved subunits together form the condensin complex. These include SMC2 and SMC4 and three non-SMC components, CAP-D2/3, CAP-G/G2, and CAP-H/H2 (Hirano and Mitchison 1994; Hirano et al. 1997). In many systems two condensin complexes have been identified, both of which contain the same SMC subunits but vary in their non-SMC components (Ono et al. 2003). In Drosophila, only one CAP-G protein has been identified; thus it likely functions in both complexes (Dej et al. 2004; Jager et al. 2005).

Some meiotic roles have been described for the condensin complex, though findings vary among systems. In Caenorhabditis elegans and Saccharomyces cerevisiae, condensin depletion or mutation results in anaphase bridging in both meiotic divisions (Hagstrom et al. 2002; Kaitna et al. 2002; Chan et al. 2004). In worms, condensin depletion does not affect SC formation or disassembly, or chromosome compaction in pachytene, though condensation is delayed later in prophase I (Chan et al. 2004). In contrast, in condensin mutants in yeast, chromosome compaction and resolution are disrupted in pachytene, and the SC fails to assemble properly (Yu and Koshland 2003).

The chromosomal passenger complex includes Aurora B kinase, INCENP, Survivin, and Borealin/Dasra. Complex members require each other for their stereotypic mitotic localization pattern in which they are found across the chromosomes in prophase, at the centromere in metaphase, and on the spindle midzone during anaphase and telophase (reviewed in Carmena and Earnshaw 2003; Vagnarelli and Earnshaw 2004). The passenger complex plays important mitotic roles in chromosome condensation, biorientation of chromatids on the spindle, chromosome separation, spindle stability, and cytokinesis. Aurora B phosphorylates many cell-cycle regulatory proteins, and the other complex members are important for its kinase activity (Kang et al. 2001; Bishop and Schumacher 2002; Honda et al. 2003). The complex has been implicated in chromosome condensation and in phosphorylation of histone H3, which correlates with condensation, although the relationship between this modification and condensation is not well understood (Gurley et al. 1978; Adams et al. 2001).

Localization of the passenger complex resembles the mitotic pattern in several meiotic systems (Parra et al. 2003; Resnick et al. 2006; Monje-Casas et al. 2007; Yu and Koshland 2007), and in both budding and fission yeast a functional similarity has been shown for the complex in destabilizing unproductive kinetochore-microtubule attachments in mitosis and meiosis (Hauf et al. 2007; Monje-Casas et al. 2007). In addition, the passenger complex plays a role in preserving centromere cohesion in meiosis I and in localizing MEI-S332/Sgo1, a protector of centromere cohesion, in both Drosophila and S. cerevisiae (Resnick et al. 2006; Monje-Casas et al. 2007; Yu and Koshland 2007). This activity is important for proper segregation of sister chromatids in meiosis II. In contrast, in C. elegans, AIR-2 (Aurora B) localizes distal to the chiasma and is required for release of arm cohesion and homolog separation in meiosis I (Kaitna et al. 2002; Rogers et al. 2002).

The related functions of the condensin and passenger complexes in chromosome condensation and resolution raise the question of whether they interact with each other. In some mitotic systems the passenger complex is required for localization (Giet and Glover 2001; Morishita et al. 2001; Hagstrom et al. 2002; Kaitna et al. 2002; Lipp et al. 2007) and phosphorylation (Lavoie et al. 2004; Lipp et al. 2007) of condensin proteins, although in other systems condensins localize independently of the passenger complex (Losada et al. 2002; Lavoie et al. 2004). The condensin complex is also suggested to be required for centromere localization of INCENP (Hudson et al. 2003).

Here we explore the meiotic roles of the condensin and passenger proteins in Drosophila oogenesis. We find that both complexes play important roles at several points in meiotic progression, but that the effects of mutations in the subunits of the two complexes are distinct from each other, in processes including SC disassembly and metaphase I chromosome configuration. We also investigate the relationship of the passenger complex and ORD, a meiotic protein with roles in chromosome condensation and cohesion (Miyazaki and Orr-Weaver 1992), and find that incenp and ord interact genetically in their regulation of metaphase I chromosome behavior.

MATERIALS AND METHODS

Fly stocks:

The dcap-gK3 and dcap-gK4 alleles and deficiency Df(2R)vg56 have been described previously (Dej et al. 2004). dcap-gZ1 and dcap-gZ2 (Z2-5052 and Z2-4027, respectively) were isolated through a genetic complementation screen with a collection of female-sterile alleles selected from a collection of nonlethal mutations (Koundakjian et al. 2004). The QA26 allele of incenp and the ord10 allele have been described previously (Bickel et al. 1997; Resnick et al. 2006). Df(2L)Exel7049 and other stocks were obtained from the Bloomington Stock Center. Flies were raised on standard Drosophila medium at 25° or 18°. Females were fattened at 25° for analysis of embryogenesis. Females were fattened at 18° for analysis of the SC, metaphase I configuration, and completion of meiosis, except dcap-g mutants were fattened at 25° for metaphase I analyses. For incenpQA26 mutants, the meiotic defects were dependent on the 18° cooler temperature.

Analysis of cytology and immunofluorescence:

Ovaries were fixed and stained as described (De Cuevas et al. 1996) with DAPI or propidium iodide for DNA, and with antibodies to C(3)G (generously provided by M. Lilly and R. S. Hawley).

Late-stage oocytes were dissected, fixed, and dechorionated between glass slides as described (Bickel et al. 2002). Oocytes were stained with TOTO-3 (Molecular Probes) and DAPI to detect DNA, and with two rat antibodies to α-tubulin (YL 1/2 and YOL 1/34, Axyll), each at 1:40 overnight. Oocytes were also stained with anti-MEI-S332 guinea pig polyclonal serum (Tang et al. 1998) at 1:1000 over 3 nights.

FISH analysis was done on stage 14 oocytes by hybridizing to the 359 satellite repeat sequence from the X chromosome and the (AATAT)6 probe for the fourth chromosome, as described (Xiang and Hawley 2006).

Embryos were collected for 30 min or 2 hr, dechorionated in 50% bleach, devitellinized in methanol and heptane, and fixed in methanol for 3 hr. Embryos were then RNase treated for 1 hr and stained with YOYO-1 (Molecular Probes) and with antibodies to α-tubulin (YL 1/2 and YOL 1/34, Axyll), each at 1:40.

In all tissues, antibodies were detected using fluorescent secondary antibodies (Jackson Immunoresesarch). Imaging of stained ovaries was performed using a Zeiss microscope with LSM510 confocal imaging software (Keck Imaging Facility), a Nikon C1 Spectral Imaging Confocal Microscope (University of Minnesota CBS Imaging Center), a Zeiss Axiophot microscope with a Spot CCD camera and software, or a Zeiss Axioskop with an AxioCam HRm camera and AxioVision AC software. Images were processed using Adobe Photoshop.

Quantification of metaphase I configuration defects and C(3)G localization was performed blind, on slides for which the genotype of the tissue had been masked.

RESULTS

Identification of female-sterile alleles of the condensin cap-g:

Alleles of the condensin dcap-g that disrupt mitotic divisions of embryogenesis and result in embryonic lethality have been previously characterized (Dej et al. 2004). We sought to identify female-sterile alleles of dcap-g that would allow us to analyze the roles of the condensin I and II complexes in the specialized cell cycles utilized in oogenesis. We screened the Zuker collection of EMS-generated, female-sterile mutations (Koundakjian et al. 2004) for mutations failing to complement the embryonic lethal allele dcap-gK1, resulting in either a lethal or sterile phenotype. We identified two mutations that were female sterile as transheterozygotes with dcap-gK1. Because only one dcap-g subunit has been identified in Drosophila, these mutations likely affect both the condensin I and II complexes.

Sequencing identified aberrations in the dcap-g sequence in both of these lines. dcap-gZ1 (Zuker mutant Z2-5052) is a point mutation predicted to change amino acid 157 from valine to glutamic acid. dcap-gZ2 (Zuker mutant Z2-4027) would convert amino acid 1215 from glutamine to a premature stop, truncating the predicted 1351-amino-acid sequence. In addition, the dcap-gK3 larval lethal allele (Dej et al. 2004) allows a few viable adult escapers, and these are also female sterile. We performed most of our analyses of condensin function, below, using the dcap-gZ1 allele transheterozygous to the dcap-gK4 allele, a putative null allele that deletes a portion of the 5′ end of the dcap-g gene (Dej et al. 2004), because this combination provided the strongest female-sterile phenotypes (data not shown). The dcap-gZ1 and dcap-gZ2 alleles are recessive for all of the phenotypes we examined, consistent with their being hypomorphic alleles that reduce protein function.

The mutations in dcap-g and incenp do not affect synaptonemal complex assembly:

We used the female-sterile alleles of dcap-g to evaluate whether, in Drosophila, condensin I or II are required for the assembly of the synaptonemal complex. We used an antibody to C(3)G, a protein in the transverse filaments of the SC. This SC protein can be visualized in the earliest oocytes, located in the germarium, in a ribbon-like structure, corresponding to the axis that has formed between the paired homologs (Page and Hawley 2001) (Figure 1A). We dissected ovaries from dcap-gZ1/dcap-gK4 females, stained with antibodies against C(3)G, and observed that the SC assembled with proper timing and morphology (Figure 1B, Table 1).

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SC assembly is normal in condensin and passenger complex mutants. In the left panels DNA is red and C(3)G is green. The middle panels show C(3)G staining and the right, DNA. (A) Oregon R control. (B) dcap-gZ1/dcap-gK4. (C) incenpQA26 Df(2L)Exel7049/incenpQA26. (D) incenpQA26 ord10/incenpQA26. All images are at the same magnification. Bar, 5μm.

TABLE 1

Synaptonemal complex assembly in condensin and passenger mutants

C(3)G localized solely to chromosome axis (%)C(3)G dispersed on axis and spreading through nucleus (%)Trace amounts C(3)G on chromosomes (%)No C(3)G associated with chromosomes (%)Na
Oregon RGerm.b100c000610 (54)d
Stage 483133030
Stage 569226332
Stage 694643335
Stage 7031432532
Stage 800386332
Stage 900118918
dcap-gZ1/dcap-gK4Germ.99.70.300631 (91)
Stage 49460017
Stage 571290014
Stage 6334720015
Stage 7165326519
Stage 8013731315
Stage 90092812
incenpQA26 Df(2L)Exel7049/incenpQA26Germ.100000104 (14)
Stage 459356017
Stage 5213636714
Stage 674047715
Stage 700326819
Stage 8013335315
Stage 90014867
incenpQA26 ord10/incenpQA26Germ.100000106 (13)
Stage 447470715
Stage 5213643014
Stage 6025552020
Stage 700534717
Stage 800297117
Stage 90022789
ord10/+Stage 490100020
Stage 568320022
Stage 693645922
Stage 7041411822
Stage 800267423
Stage 90089224

Quantification of C(3)G localization was performed blind for all genotypes except the ord10/+ experiment. Germ., germarium.

aN, number of oocytes scored.
bC(3)G staining was scored in regions 2a, 2b, and 3 of the germarium.
cNumbers over 30% are boldface as a visual aid to highlight the behavior of the preponderance of the oocytes.
dIn these samples the number of nuclei with C(3)G staining is shown and in parenthesis the number of germaria scored is given.

We similarly used the QA26 female-sterile allele of incenp to test for effects on SC loading. Because the incenpQA26 allele is weak and resulted in only modest meiotic defects on its own, we sought to enhance its effects in female meiosis. incenpQA26 generates a single-amino-acid change in the C-terminal IN-BOX of the INCENP protein, the region through which INCENP interacts with Aurora B kinase (Adams et al. 2000; Resnick et al. 2006). We reasoned that the mutation likely weakens the interaction between these two proteins, and that reducing the amount of Aurora B protein might enhance the meiotic phenotype. We crossed one copy of the small chromosomal deficiency Df(2L)Exel7049, which removes 18 genes including Aurora B (Parks et al. 2004), into the incenpQA26 background.

We dissected ovaries from incenpQA26 Df(2L)Exel7049/incenpQA26 and wild-type flies, and we stained for C(3)G. In these mutants as well, assembly of the SC was unaffected (Figure 1C, Table 1).

The mutations in dcap-g and incenp differentially affect synaptonemal complex maintenance:

The SC is present only transiently in prophase I, and its disassembly can be visualized by a gradual and progressive dissociation of C(3)G staining from the chromosomes. Developing egg chambers progress through distinguishable stages that have been defined by their morphological characteristics, beginning at stage 2, after the egg chamber has left the germarium, and continuing until stage 14, at which point the egg is mature. The SC disassembles gradually, beginning at stage 4 of egg chamber development and is complete by stage 9 (Table 1). We defined the phases of disassembly cytologically as follows: (1) assembled SC shows C(3)G staining solely on the chromosome axis (Figure 2A); (2) initial phases of SC disassembly are marked by C(3)G being more dispersed on the chromosome axis and the presence of the antigen in the oocyte nucleus (Figure 2B); (3) as the SC further disassembles C(3)G present only in trace amounts on the chromosomes (Figure 2C); and (4) finally no C(3)G is detectable on the chromosomes but the antigen in present throughout the oocyte nucleus (Figure 2D).

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Developmental timing of SC disassembly. In the left panels DNA is red and C(3)G green; side panels show separated channels for boxed region, with C(3)G on the top. In wild-type oocytes C(3)G localizes to the meiotic chromosomes beginning in the germarium, prior to stage 2. This localization to the chromosomes, which are in a compact structure and take up only a small portion of the nucleus, is maintained through stage 4 or 5. As development continues, C(3)G begins to delocalize from the chromosomes and is seen with increasing intensity throughout the nucleus. (A–D) Display of the categories scored in Table 1. (A) C(3)G localized solely to chromosome axis (Oregon R, stage 4). (B) C(3)G dispersed on axis and spreading through nucleus (Oregon R, stage 6). (C) Trace amounts of C(3)G on chromosomes (Oregon R, stage 6). (D) No C(3)G associated with chromosomes (Oregon R, stage 8). All of the merged images are at the same magnification. Bar, 10μm.

Loading of the condensin complex in meiotic prophase I correlates temporally with the disassembly of the synaptonemal complex (Ivanovska et al. 2005; Ivanovska and Orr-Weaver 2006). A suggestion that there is an important regulatory link between these two processes, as well as the temporal correlation, comes from mutation of nucleosomal histone kinase-1 (nhk-1), which disrupts both condensin loading and SC unloading (Ivanovska et al. 2005). We used the dcap-g alleles to examine whether function of the condensin complex on the chromosomes is required for unloading of synaptonemal complex proteins.

A clear delay in disassembly of the SC in the dcap-g mutant was observed following quantification of the number of oocytes in stages 4–9 in each of the four phases of SC disassembly (Table 1). In wild-type ovaries, C(3)G was associated solely with the chromosome axis in only 9% of stage 6 egg chambers, compared with 33% of dcap-g ovaries at the same stage. By stage 7, none of the wild-type oocytes had C(3)G solely on the axis, and only 31% of wild-type oocytes displayed C(3)G dispersed along the axis and spreading through the nucleus. In contrast, 16% of dcap-g oocytes at stage 7 showed C(3)G to be solely chromosome localized, and another 53% at the same stage displayed C(3)G dispersed along the axis. Furthermore, in wild-type oocytes, most had no remaining C(3)G on the chromosomes by stage 8 and the vast majority showed no chromosome-specific C(3)G in stage 9. dcap-g mutants retained at least trace levels of C(3)G on the chromosomes in nearly all oocytes at these stages. Figure 3 illustrates C(3)G localization in dcap-g mutants and its persistence into stage 8 on the chromosomes. We used Fisher's exact test to determine whether the frequency of stage 7 oocytes with C(3)G fully or mostly on the chromosomes vs. in trace amounts or undetectable on the chromosomes was significantly different between wild-type and dcap-g mutants and found it was, with P = 0.02. Thus we conclude that the condensin complex is indeed required for proper disassembly of the synaptonemal complex. Furthermore, because the dcap-g mutations are weak alleles, the condensin complex likely plays an even more important role in SC disassembly than is evidenced by the delay seen here.

An external file that holds a picture, illustration, etc. Object name is GEN1813875f3.jpg

SC dynamics and delayed disassembly in dcap-g mutants. SC structure in progressive stages of oocyte development in dcap-gK3/Df mutant oocytes. DNA is in green; C(3)G is in red in the merged images and in white in the insets. (A) In the germarium, SC assembles normally in the dcap-g mutant. (B and C) The SC is present during the pachytene stages, as in wild-type oocytes. (D) The SC aberrantly persists into stage 8. The developmental stage is shown in each panel. The merged images are at the same magnification. Bar, 20 μm; bars, 5 μm in all the enlarged insets.

We explored the role of the passenger complex in SC disassembly by quantifying C(3)G staining patterns in the incenpQA26 Df(2L)Exel7049/incenpQA26 mutant. Strikingly, we found that, rather than prolonged maintenance of the SC, these mutants displayed premature disassembly of this structure (Table 1). In the wild-type control, C(3)G was localized solely to the chromosome axes in 69% of stage 5 oocytes, and only 6% of stage 5 oocytes showed C(3)G spread throughout the nucleus with just trace amounts of C(3)G associated specifically with the DNA. In incenpQA26 Df(2L)Exel7049/incenpQA26 ovaries, disassembly of the SC was well underway in stage 5, with only 21% of oocytes displaying C(3)G strongly associated with the chromosomes and 36% showing only trace amounts of C(3)G remaining specifically localized to the DNA (premature disassembly shown in Figure 4). By Fisher's exact test the percentages of stage 5 oocytes with full or partial SC on the chromosomes vs. in trace amounts or undetectable levels are significantly different between the incenpQA26 Df(2L)Exel7049/incenpQA26 and Oregon R controls (P = 0.02). Thus, the mutation of two protein complexes that are both involved in chromosome condensation has different effects on SC disassembly in meiosis. The results are consistent with the chromosome passenger complex being required for maintenance of the synaptonemal complex and the condensin complex playing an important role in its disassembly.

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The SC prematurely disassembles in incenp mutants. The SC is disassembled completely in this stage 5 oocyte from an incenpQA26 Df(2L)Exel7049/incenpQA26 female, because there is no detectable C(3)G staining on the chromosomes. The DNA is shown in red and C(3)G in green in the merged image on the left, and split channels are shown as labeled on the right. Bar in the merged image, 5 μm.

The condensin complex is required for metaphase I arrest:

We next analyzed the roles of the condensin complex in the metaphase I chromosome configuration. The stable biorientation of homologs in metaphase I, which is critical for proper segregation of the homologs in anaphase I, is very different from mitotic chromosome behavior (Petronczki et al. 2003). In many systems, including Drosophila female meiosis, this metaphase I configuration is further distinguished from mitosis because the spindle lacks centrosomes (Theurkauf and Hawley 1992). The female meiotic spindle is organized by the chromosomes themselves, as they capture microtubules that are bundled into poles by kinesin-like motor activity. In Drosophila female meiosis, stages 12 and 13 correspond to prometaphase I and follow egg maturation. The mature stage 14 egg is arrested in metaphase I until ovulation (Mahowald et al. 1983). During stages 12 and 13 the achiasmate chromosomes are often positioned between the spindle poles and the main chromosome mass, but by stage 14 the nonexchange chromosomes retract into the main chromosome mass, which is tightly repackaged with properly cooriented centromeres (Gilliland et al. 2007, 2008).

To ask whether the condensin complex is involved in the establishment or maintenance of the metaphase I configuration, we stained ovaries to visualize the DNA, and we examined the chromosome morphoplogy in late-stage mutant oocytes. We distinguished between prometaphase I oocytes (stages 12 and 13) and metaphase I oocytes (stage 14) by the presence or absence of nurse cell debris, respectively. Nurse cells are large, polyploid cells that produce vast quantities of mRNA and protein, then undergo apoptosis and dump these products into the oocyte in stage 11 of development. By stage 14, nurse cell debris is eliminated (Spradling 1993).

In wild-type oocytes that had exited the prophase I arrest, the chiasmate chromosomes were predominantly seen in a single, round, compact mass. The chromosomes were elongated or separated into multiple chromosome masses in <20% of oocytes. This was true both in prometaphase I and metaphase I oocytes (Table 2). In dcap-gZ1/dcap-gK4 mutants, stage 12 and 13 oocytes showed a modest increase in the number of prometaphase I configurations with separated chromosomes. Stage 14 oocytes revealed a dramatic increase in metaphase I defects, most commonly multiple chromosome masses (Table 2, Figure 5, A and B). This is a recessive phenotype (Table 2).

TABLE 2

Mutations in dcap-g and incenp prevent proper organization of metaphase I configuration

Stages 12 and 13 oocytes (nurse cell debris visible)
Stage 14 oocytes (no nurse cell debris present)
% prometaphase I with separated large chromosomesNa% aberrant metaphase I configurationsN
w11181729978
dcap-gZ1/dcap-gK426154520
dcap-gZ1/+NDb1030
dcap-gK4/+ND042
incenpQA26/incenpQA265549946
incenpQA26 Df(2L)Exel7049/incenpQA26694917115
incenpQA26 ord10/incenpQA2685205343

Quantification of prometaphase I or metaphase I configuration as determined by DAPI staining was performed blind, except for the dcap-gZ1/+ and dcap-gK4/+ experiments.

aN, number of oocytes scored.
bND, not scored.
An external file that holds a picture, illustration, etc. Object name is GEN1813875f5.jpg

dcap-g mutant oocytes have defective metaphase I figures. (A) FISH analysis shows that in the w1118 control the chromosomes are held on the metaphase I plate, with the X chromosomes (green FISH signal) bioriented. Bar, 3 μm. (B) In dcap-gZ1/K4 mutant mature oocytes multiple chromosome masses are seen at metaphase I. The FISH probes (X, green; fourth, red) show that the homologs are not only bioriented but separated. Bar, 2 μm. (C) MEI-S332 (red) localization to the centromeres of a control Oregon R oocyte. The spindle is shown in green. (D–F) MEI-S332 localizes to the centromeres in dcap-g mutants. In all panels DNA stain is in blue. The images in C–F are at the same magnification. Bar, 5 μm.

We used fluorescent in situ hybridization (FISH) analysis with probes specific for the X and fourth chromosomes to test whether premature sister-chromatid separation occurred in the dcap-g mutants and whether the X homologs were separated. (The fourth chromosomes normally separate and move to opposite poles during prometaphase I in wild type.) We did not observe premature sister separation, but the X homologs and autosomes did prematurely disjoin from their bivalants in the dcap-g mutants (Figure 5, A and B). (0/75 stage 14 had separated X bivalents in the w1118 control compared to 4/15 in dcap-gZ1/dcap-gK4 oocytes, plus an additional oocyte with separated autosomes but an intact X bivalent. These are significantly different by Fisher's exact test, P = 5 × 10−4).

We examined the spindle morphology and centromere orientation of meiotic chromosomes in stage 12–14 oocytes by staining with antibodies to tubulin and the centromere protein MEI-S332 (Kerrebrock et al. 1995). In wild-type oocytes, most metaphase I figures displayed the normal morphology in which a single chromosome mass was located at the center of a thin, tapered spindle, and centromeres were positioned toward each spindle pole, reflecting stable biorientation on the spindle (Figure 5C) (Moore et al. 1998). We observed that MEI-S332 is localized in the dcap-g mutants (Figure 5, D–F) and found examples of single univalents migrating to the pole (4/9 oocytes) (Figure 5F). These observations are consistent with the conclusions from the FISH experiment that sister-chromatid cohesion is retained in dcap-g mutants but that bivalents prematurely separate into homologs by metaphase I.

The passenger complex is required for biorientation of bivalents in prometaphase I:

DAPI staining revealed that over half the stage 12 and 13 incenpQA26 oocytes displayed prometaphase I configurations with separated chromosomes, but that stage 14 oocytes of the same genotype exhibited configurations comparable to those seen in wild-type mature oocytes (Table 2). incenpQA26 Df(2L)Exel7049/incenpQA26 oocytes showed a similar pattern: 69% of stage 12 and 13 oocytes contained prometaphase I with multiple chromosome masses, whereas only 17% of stage 14 oocytes displayed aberrant metaphase I chromosome configuration. These results are striking because premature loss of cohesion, as observed in incenpQA26 male meiosis (Resnick et al. 2006), would not explain a defect in which chromosome masses separated from each other transiently and then congressed to a single, stable chromosome mass.

We performed FISH experiments to define the X and fourth chromosome configurations in metaphase I oocytes from incenpQA26 Df(2L)Exel7049/incenpQA26 mutant females (Figure 6, A–C). We did not observe premature separation of the sister chromatids. In striking contrast to the w1118 control, however, the two X chromosomes frequently failed to biorient and were oriented toward the same pole (0/75 for w1118 and 8/10 for incenpQA26 Df(2L)Exel7049/incenpQA26. These are significantly different by Fisher's exact test, P = 9 × 10−10) (Figure 6, A–C). This biorientation failure accounts for the extra chromosome masses observed in prometaphase I, when a monooriented bivalent would have congression defects. The spindle shortens in the transition from prometaphase I to metaphase I (Gilliland et al. 2007, 2008), and this may contribute to pushing even monooriented bivalents to the metaphase plate and the apparent recovery we observe in stage 14. The fourth chromosomes most often bioriented, and separated univalents were observed toward each spindle pole (Figure 6, B and C), but we sometimes also saw the pair of fourth chromosomes monooriented.

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Biorientation and segregation failure in incenp mutant oocytes. (A–C) FISH analysis of chromosome orientation. In contrast to the biorientation observed for the X bivalent (green FISH signal) in the w1118 control (A), in incenpQA26 Df(2L)Exel7049/incenpQA26 mutant oocytes the X bivalent (green) is monooriented (B and C). In these oocytes the fourth chromosomes (red) are bioriented. (D and E) Staining for MEI-S332 (red), spindle (green), and DNA (blue). MEI-S332 localizes to chromosomes in incenp mutant oocytes. Although it can localize in a pattern consistent with centromere localization (arrow in D), extra foci frequently are observed (E). (F and G) FISH analysis of chromosomes. Bivalents fail to biorient in incenpQA26 ord10/incenpQA26 oocytes. In F, the X chromosomes are bioriented (green) but the fourth chromosomes (red) monooriented, whereas the opposite is observed in G. Bars are indicated in each panel.

In male meiosis incenp function is required for the proper centromere localization of MEI-S332 (Resnick et al. 2006). In incenp mutant oocytes, however, we observed MEI-S332 with apparent centromere localization (Figure 6D, arrow), presumably due to sufficient passenger complex activity in this tissue. incenpQA26 spermatocytes display MEI-S332 across the chromatin in a diffuse pattern (Resnick et al. 2006). In incenpQA26 Df(2L)Exel7049/incenpQA26 oocytes we frequently observed more than the eight foci predicted for localization to each of the paired sister centromeres (Figure 6, D and E). Thus it is likely that passenger complex function is required for restricted localization of MEI-S332 to the centromeres in oocytes as well as spermatocytes. We cannot exclude the possibility that some premature loss of sister-chromatid cohesion occurs in these mutant oocytes, possibly not detected by FISH due to the biorientation failure.

Mutation of the meiotic gene ord dominantly enhances incenpQA26:

The gene ord has also been shown to have an important role in meiotic chromosome condensation. In male meiosis, mutation of ord results in defects in packing and pairing of the prophase I bivalents that are remarkably similar to the phenotypes observed in incenpQA26 spermatocytes (Miyazaki and Orr-Weaver 1992; Resnick et al. 2006). Furthermore, just as the passenger complex is required for synaptonemal complex maintenance, so too is ORD (Webber et al. 2004). Therefore, we asked whether ord and incenp interact genetically. We introduced a single copy of the ord10 allele into an incenpQA26 background. ord10 generates an early stop codon, and therefore is presumed to be a null allele (Bickel et al. 1997). ord10 heterozygotes do not display metaphase I defects on their own (Bickel et al. 2002).

We stained the DNA of incenpQA26 ord10/incenpQA26 ovaries and examined late-stage oocytes. Eighty-five percent of stage 12 and 13 oocytes displayed prometaphase I configurations with separated chromosome masses, a significant increase over the 55% seen in incenpQA26 mutants, P = 0.03 (Table 2). Even more strikingly, over half the stage 14 incenpQA26 ord10/incenpQA26 oocytes also showed metaphase I chromosome defects, in contrast to resolution of these defects seen by this stage in incenpQA26 alone. (By Fisher's exact test these are significantly different, P = 6 × 10−6.) FISH analysis with the X and fourth chromosome probes showed these oocytes also exhibited a failure of bivalent biorientation (6/7 oocytes) (Figure 6, F and G). These observations raise the possibility that when levels of ORD are reduced, in addition to the failure of bivalents to biorient, another defect may be manifest that hinders these monooriented bivalents from being pushed into a metaphase I configuration. The enhanced phenotype shows that ord and incenp interact genetically and that both are involved in metaphase I chromosome dynamics.

In addition to the metaphase I defect, incenpQA26 ord10/incenpQA26 females also exhibited premature disassembly of the synaptonemal complex, at frequencies comparable to the incenpQA26 Df(2L)Exel7049/incenpQA26 oocytes (Table 1). Assembly of the synaptonemal complex in this mutant combination is indistinguishable from wild type (Figure 1D, Table 1). The ord10 mutation has no dominant effect on the synaptonemal complex, because ord10/+ oocytes were stained with anti-C(3)G and the assembly and disassembly were no different from wild type (Table 1).

dcap-g and incenp mutant females produce embryos that arrest with mitotic defects:

Failure to biorient bivalents in meiosis would lead to improper chromosome segregation, and this appears to be the case in embryos from incenpQA26 mutant mothers. The meiotic products frequently are of unequal size, indicative of unequal numbers of chromosomes (data not shown). The female sterility observed in the dcap-g and incenp mutants is the consequence of early arrest in embryos produced by these mutant mothers. These embryos underwent at most three mitotic divisions, with defects such as polyploid chromosome number and anaphase bridging evident (Figure 7, A–D). The condensed chromosomes normally present in the polar body rosette structure had aberrant morphologies in embryos from dcap-g or incenp mutant mothers (Figure 7, E–H). Thus the meiotic defects, likely coupled with mitotic defects in early embryos due to the absence of functional maternal pools of the condensin and chromosome passenger complex, result in early embryonic lethality.

An external file that holds a picture, illustration, etc. Object name is GEN1813875f7.jpg

dcap-g and incenp mutant mothers produce embryos displaying defects in mitosis and postmeiotic chromosome structure. Early embryos stained for DNA (green, and gray) and tubulin (red). (A) Oregon R, anaphase in first mitotic division. (B) A dcap-gZ1/dcap-gK4, polyploid mass in early arrested embryo. (C) incenpQA26, chromosome bridging in first anaphase. (D) incenpQA26, chromatin bridging in early arrested embryo. (E–H) polar body rosette structures. (E) Oregon R, displays condensed arms of even length. (F) dcap-gK3, pulverized chromosomes. (G) dcap-gZ1, hypercondensed chromosomes. (H) incenpQA26, elongated and fragmented chromosomes. The images in A–D are at the same magnification. Bars, 5 μm.

DISCUSSION

The condensin and chromosomal passenger complexes both have important roles in chromosome condensation in mitosis, and the passenger complex has been shown in many systems to be required for localization or phosphorylation of condensin proteins (Giet and Glover 2001; Morishita et al. 2001; Hagstrom et al. 2002; Kaitna et al. 2002; Lavoie et al. 2004; Lipp et al. 2007). Here we observe distinct meiotic consequences of mutations in dcap-g and incenp. Strikingly, SC disassembly was premature in incenp mutants but delayed in dcap-g mutants, and prometaphase I and metaphase I chromosome configurations were disrupted in both mutants, but in clearly distinguishable ways.

That both the condensin and passenger complexes affect SC disassembly is intriguing because little is known about regulation of this process. BubR1 has recently been shown to be required for SC maintenance (Malmanche et al. 2007), although the mechanism has not yet been established. A suggestion that condensin might be required for SC disassembly arose from the observation that mutation of nhk-1 disrupts both condensin loading and C(3)G unloading from the chromosomes, within the same developmental window (Ivanovska et al. 2005). In the germarium, multiple cells within each cyst initiate meiosis and form SC but ultimately the SC disassembles in all the cells except the oocyte (Spradling 1993; Page and Hawley 2001). Interestingly, the dcap-g mutations affect SC disassembly solely in the oocyte, not in the nurse cells at the earlier developmental stage. This corresponds to when condensin assembles on the oocyte chromosomes and raises the possibility of a distinct mechanism of SC disassembly in the nurse cells. Condensin seems not to be required for SC assembly or disassembly in C. elegans, whereas it is required for proper SC assembly in S. cerevisiae (Yu and Koshland 2003; Chan et al. 2004). Understanding the differences among these systems and the manner in which condensin is required for SC disassembly in Drosophila remain important questions for future study.

A requirement for the passenger complex in maintenance of the SC is striking in combination with the result that incenp and ord interact genetically. ord is required, as well, for SC maintenance (Webber et al. 2004). incenp could regulate ord, and the SC phenotype seen in the incenp mutant could be due to defects in ORD localization or activity. In addition, SC disassembly in C. elegans has been suggested to play a role in positioning AIR-2 (Aurora B) for its role in releasing cohesion at the onset of anaphase I (Nabeshima et al. 2005), suggesting that these two complexes might dynamically regulate each other's localization.

The defects in prometaphase I and metaphase I configurations lead to several important conclusions about the functions of the passenger and condensin complexes. First, mutations in both complexes resulted in clear abnormalities, strongly supporting roles for both in a stable metaphase I chromosome configuration. Second, the defects from the dcap-g and incenp mutations were different from each other, suggesting that their roles are distinct and that the phenotypes observed in the incenp mutants are not mediated by defects in condensin localization or activity. Third, ord dominantly enhances the incenpQA26 mutation. Taken together with the observations that ord10 does not display dominant behavior alone or in a mei-S332 background (Bickel et al. 1998), this enhancement of incenp may reveal an important functional relationship between the two proteins.

In the dcap-g mutants bivalents biorient, and sister chromatids are not prematurely separated, but in metaphase I the homologous chromosomes frequently prematurely separate and begin poleward migration. One explanation for this phenotype is that the chiasmata, which normally provide the physical linkage between the homologs in the bivalent, are not present. This could be due either to an absence of recombination, a failure to form chiasmata, or the premature loss of chiasmata. The latter two effects could result from loss of arm cohesion. Given the female sterility of the dcap-g alleles, it is not possible to recover viable progeny to measure recombination frequencies. Thus currently it is difficult to distinguish between these possibilities. In addition to the distinct functions of condensin in the dynamics of the synaptonemal complex in different organisms, our results highlight differences in the role of condensin in homolog attachments. In Drosophila males, in which recombination does not occur, the condensin II complex is required for homolog separation in anaphase I (Hartl et al. 2008). In budding yeast condensin is needed to resolve recombination linkages between homologs by promoting the release of cohesin (Yu and Koshland 2003). In C. elegans, the dpy28 gene that encodes a Cap-D2 protein blocks recombination and affects interference, although it is not yet clear whether it has this function as part of a condensin complex (Tsai et al. 2008).

The incenpQA26 Df(2L)Exel7049/incenpQA26 mutants show a high frequency of failure of bivalents to biorient. This result indicates that in metazoan meiosis, as in budding and fission yeast, the chromosome passenger complex and Aurora B are likely to have the ability to destabilize improper microtubule kinetochore attachments. Homolog monoorientation is observed in meiosis I in yeast mutant for Aurora B (Hauf et al. 2007; Monje-Casas et al. 2007). The biorientation failure explains the multiple chromosome masses observed in prometaphase I, although it is surprising that the monooriented bivalents nevertheless are in a normal compacted chromosome mass by metaphase I. It most likely results from the shortening of the spindle that occurs in the transition from prometaphase I to metaphase I (Gilliland et al. 2007, 2008). While this work was under review Colombie et al. (2008) reported that incenpQA26 mutant oocytes have spindle defects. They found that the assembly time for the spindle was extended in prometaphase I and that the central spindle region was less stable in metaphase I. This effect on the metaphase I spindle stability correlates with the observation that INCENP protein localizes to the spindle midregion (Jang et al. 2005). The aberrant bivalent orientation we observed raises the possibility that the chromosome defects in incenpQA26 are responsible for the delayed bipolar spindle assembly and metaphase I spindle instability, given that the chromosomes organize this acentriolar spindle.

It is important to note that incenpQA26 exhibits somewhat distinct effects in male and female meiosis. In male meiosis in these mutants chromosome condensation is defective and premature loss of sister-chromatid cohesion occurs, but the bivalents biorient. In contrast, in female meiosis in the mutants sister-chromatid cohesion is not lost, but bivalent orientation and spindle formation are defective. These phenotypes may result from important regulatory differences in male and female meiosis, indeed the differing mechanisms of meiotic spindle formation necessitate variations in chromosome-spindle interactions. Alternatively, the distinct phenotypes could arise due to the hypomorphic incenpQA26 allele differentially affecting passenger complex activity in the two tissues.

The identification of female-sterile alleles of dcap-g and incenp has led to the demonstration of critical roles for these proteins, and presumably the protein complexes in which they participate, in several critical aspects of chromosome dynamics during oocyte meiosis. These mutants point to distinct roles for the condensin and chromosome passenger complexes in control of the synaptonemal complex, and they illustrate the crucial role of each of these complexes in formation of a stable metaphase I configuration.

Acknowledgments

We are grateful to Mousumi Mutsuddi for the ord10/+ control C(3)G staining and for preparing the dcap-gZ1, Z2, and K4 heterozygous control oocytes and embryos. We thank Barbara Wakimoto, Dan Lindsley, and Mike McKeown who screened the Zuker mutant collection for female-sterile lines, George Bell for assistance with statistical analysis, Janice Lee for helpful discussions, Mary Lilly for antibodies, Tom Dicesare for help with figure preparation, and Andreas Hochwagen, Cintia Hongay, and Laura Lee for critical comments on the manuscript. Thanks to Hiroyuki Ohkura for sharing in-press results. Some of the microscope images were collected in the Keck Imaging Facility of the Whitehead Institute or in the University of Minnesota CBS Imaging Center. T.D.R. was supported by an Anna Fuller graduate fellowship. R.S.H and T.O.-W. are American Cancer Society Research Professors. This research was supported by National Science Foundation grant MCB0132237 and National Institutes of Health grant GM39341 to T.O.-W. Support for some aspects of this work was provided by the Stowers Institute for Medical Research.

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