Decision letter: The asymmetry of female meiosis reduces the frequency of inheritance of unpaired chromosomes

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Trisomy, the presence of a third copy of one chromosome, is deleterious and results in inviable or defective progeny if passed through the germ line. Random segregation of an extra chromosome is predicted to result in a high frequency of trisomic offspring from a trisomic parent. Caenorhabditis elegans with trisomy of the X chromosome, however, have far fewer trisomic offspring than expected. We found that the extra X chromosome was preferentially eliminated during anaphase I of female meiosis. We utilized a mutant with a specific defect in pairing of the X chromosome as a model to investigate the apparent bias against univalent inheritance. First, univalents lagged during anaphase I and their movement was biased toward the cortex and future polar body. Second, late-lagging univalents were frequently captured by the ingressing polar body contractile ring. The asymmetry of female meiosis can thus partially correct pre-existing trisomy. https://doi.org/10.7554/eLife.06056.001 eLife digest Inside cells, DNA is found packaged into structures called chromosomes. Most human and animal cells contain two sets of chromosomes, one inherited from each parent. Chromosomes from one set pair up with the equivalent chromosome from the other set. However, egg and sperm cells only contain one copy of each chromosome, so that when the egg is fertilized, the resulting cell again has two sets of chromosomes. If there are either more or fewer than two copies of a chromosome in the fertilized cell, this can cause birth defects and conditions such as Down syndrome. An egg cell develops from a cell called an oocyte via a process called meiosis. The oocyte first duplicates its DNA so that it contains four copies of each chromosome. The oocyte then divides, and the resulting cells divide again, to produce four cells that each contains one copy of each chromosome. Only one of these cells is an egg cell: the other three are called polar bodies, and these normally self-destruct. The tiny roundworm C. elegans is a model organism used to study meiosis. Worms can be hermaphrodites or males; the hermaphrodites normally have a pair of ‘X’ sex chromosomes. However, sometimes problems with meiosis can produce hermaphrodite worms with three X chromosomes in each of their cells. In these cells, two of the X chromosomes pair with each other as normal, and one X chromosome remains unpaired. Cortes et al. examined meiosis in mutant worms that had an extra copy of the X chromosome by marking all the chromosomes with a fluorescent tag. This allowed the movement of the chromosomes to be tracked through images taken using a microscope. This revealed that an unpaired X chromosome moves more slowly than a normal paired set. Furthermore, the unpaired chromosomes tend to move toward the region of the oocyte that will develop into a polar body. Thus, when the oocyte divides, the unpaired chromosomes are placed in the polar body and eliminated. This mechanism improves the chance that the correct number of chromosomes will end up in the egg cell. Women with three X chromosomes are often fertile and in most cases produce normal offspring. Further work is needed to see whether human oocytes remove extra chromosomes by a mechanism similar to that seen in the roundworms. https://doi.org/10.7554/eLife.06056.002 Introduction During female meiosis, a G2 oocyte containing four genome copies undergoes two asymmetric cell divisions depositing one genome in a single haploid egg, while the other three genomes are segregated into polar bodies. These divisions are mediated by meiotic spindles that are asymmetrically positioned against the oocyte cortex with the pole-to-pole axis of the spindle perpendicular to the cortex. Both the inheritance of only one of the four genome copies and the distinct perpendicular positioning of the meiotic spindle are remarkably conserved among animal phyla suggesting a selective advantage (Maro and Verlhac, 2002; Fabritius et al., 2011a; Maddox et al., 2012). Several advantages of asymmetric meiosis have been suggested previously, yet none are applicable to all animals. Asymmetric meiotic spindle positioning maximizes the volume of a single egg, helps prevent interference with the meiotic spindle by the sperm aster (McNally et al., 2012), and preserves predetermined embryonic polarity gradients. Here, we suggest a previously unrecognized advantage of asymmetric meiosis, the ability of meiotic spindles to correct trisomy by preferentially depositing the extra chromosome copy into a polar body. Accurate segregation of homologous chromosomes to opposite spindle poles depends on a physical attachment, or chiasma, between homologous chromosomes. A chiasma consists of a crossover, which holds the two homologous chromosomes together in a bivalent so that kinetochores can be properly oriented toward opposite poles (Moore and Orr-Weaver, 1998; Miller et al., 2013). When a chiasma does not form, univalent chromosomes may maintain sister cohesion and move to poles independent of their homologs at anaphase I as can occur in Saccharomyces cerevisiae (Buonomo et al., 2000). If a univalent chromosome biorients, loses cohesion, and segregates sister chromatids at anaphase I (e.g., Nicklas and Jones, 1977; Lemaire-Adkins and Hunt, 2000; Kouznetsova et al., 2007), the resulting single chromatid will segregate randomly at anaphase II. Random segregation of homologs at anaphase I or single chromatids at anaphase II should result in equal frequencies of haplo and diplo ova in the case of a trisomy (Figure 1A) and equal frequencies of nullo and diplo ova in the case of a crossover failure. Figure 1 Download asset Open asset Trisomy correction during meiosis I. (A) Illustration showing expected outcomes of female meiosis in XXX wild-type worms, assuming the extra univalent X (red) does not lose cohesion (yellow) between sister chromatids during anaphase I and assuming random segregation. (B) Illustration of a spindle with chromosomes at the metaphase plate with poles marked ‘P’ (left) and a projection of the cross-sectional view down the pole-to-pole axis at the metaphase plate (right). (C–E) Z projections of fixed meiotic embryos viewed down the pole-to-pole spindle axis. Meiotic embryos from XXX wild-type mothers were stained with DAPI and anti-tubulin antibody. (C) Metaphase I spindles with 7 chromosomes; right images of X-fluorescence in situ hybridization (FISH) show two X chromosomes on the spindle. See also Supplementary file 2. (D) Metaphase II spindles with 6 chromosomes; right images of X-FISH show one X on the spindle and 2–3 foci in the polar body. See also Supplementary file 3. (E) Metaphase II spindle with 7 chromosomes. (F) Frequency of each spindle class among the progeny of XXX wild-type mothers. Insets show polar bodies, marked by asterisks, which were used to identify metaphase II spindles. Bar = 5 μm. https://doi.org/10.7554/eLife.06056.003 Deviations from random segregation are suggested by observations of X chromosomes in Caenorhabditis elegans. In C. elegans, the single unpaired X chromosome from an XXX mother is inherited with unexpectedly low frequency with twice as many haploX ova produced as diploX ova (Hodgkin et al., 1979). HIM-8 is a zinc finger protein that binds to specific DNA sequences that are enriched on the X chromosome. him-8 mutants have a pairing defect that is completely specific for the X chromosome, resulting in two X univalents and five autosomal bivalents in 95% of diakinesis oocytes (Phillips et al., 2005). If the two X univalents segregated randomly, him-8 mutants would be expected to produce equal frequencies of nulloX ova and diploX ova. However, Hodgkin et al. (1979) demonstrated a fivefold preponderance of nulloX ova over diploX ova in him-8. Using sex-reversed him-8 XX males, these authors showed the opposite effect in spermatogenesis. Rather than producing the 50% haploX, 25% diploX, 25% nulloX sperm expected from random segregation, him-8 XX males produced 86% haploX, 3% diploX, 11% nulloX sperm, indicating symmetric distribution of univalents during male meiosis. Thus, achiasmate maternal X chromosomes are inherited with unexpectedly low frequency in worms. Five mechanisms might reduce the frequency of trisomic offspring from trisomic or him-8 mothers. First, trisomic embryos might die during embryonic development resulting in undercounting of XXX offspring. This is unlikely in C. elegans because both XXX and him-8 mutant mothers produce a very low frequency of dead embryos (Hodgkin et al., 1979; Supplementary file 1). A second possibility is that mitotic non-disjunction in the XXX mother results in a mosaic gonad that contains both diploX and triploX oocytes. Selective apoptosis of XXX germ line cells (Bhalla and Dernburg, 2005) would then enrich for XX germ line cells. This does not contribute to the segregation bias in C. elegans, as the most mature diakinesis oocytes in him-8 and wild-type XXX worms have 7 rather than 6 DAPI-staining bodies (Phillips et al., 2005; this study). A third possibility is that a univalent present during metaphase I or a single chromatid present during metaphase II would be broken or otherwise degraded during anaphase. A fourth possibility is that many XXX progeny look normal because of the stochastic nature of dosage compensation and thus are undercounted. A fifth possibility is that univalent chromosomes present at metaphase I are preferentially placed in the first polar body. Here, we demonstrate that indeed biased deposition of univalent X chromosomes into the first polar body reduces the frequency of trisomic zygotes resulting from oocytes with unpaired X chromosomes. Results XXX wild-type oocytes preferentially lose the achiasmate X chromosome between metaphase I and metaphase II Elimination of the extra chromosome from an oocyte starting with a trisomy would result in rescue to a euploid state. It has previously been shown that C. elegans XXX wild-type oocytes have a paired bivalent X and an unpaired univalent X chromosome in pachytene (Goldstein, 1984). We picked wild-type XXX adult hermaphrodites from the progeny of an XXX strain (AV494, Mlynarczyk-Evans et al., 2013) based on their characteristic dumpy morphology as described by Hodgkin et al. (1979). Meiotic embryos from XXX mothers were fixed and stained for microtubules and DNA. Chromosomes are well separated by bundles of microtubules during C. elegans female meiotic metaphase. This unique morphology facilitates counting of individual chromosomes on the metaphase plate when viewed down the pole-to-pole axis of the spindle (Figure 1B). We found that 100% of metaphase I meiotic embryos from XXX wild-type worms had 7 DAPI-staining bodies on the spindle (Figure 1C,F), consistent with 6 bivalents and a single univalent X. Two chromosomes were labeled with an X-specific fluorescence in situ hybridization (FISH) probe in these spindles (Figure 1C, Supplementary file 2). This result shows that a mosaic gonad resulting from mitotic nondisjunction cannot explain the low frequency of XXX offspring from XXX worms. If the univalent segregated randomly during anaphase I, 50% of metaphase II spindles should have 6 DAPI-staining bodies (6 bivalents) and 50% should have 7 DAPI-staining bodies (6 bivalents and 1 univalent). Instead, 71% of metaphase II spindles contained only 6 DAPI-staining bodies and only 29% contained 7 DAPI-staining bodies (Figure 1D–F). These frequencies match the 2:1 ratio of X to XX ova previously interpreted from genetic studies (Hodgkin et al., 1979) and are significantly different than the 50% expected from random segregation (one-tailed p = 0.004, Pearson's chi-squared test). This result eliminates the possibilities that XXX mothers have many XXX offspring that are undercounted due to incomplete penetrance of the XXX dumpy phenotype or that hermaphrodite nulloX sperm contributes significantly to the low frequency of XXX self-progeny. The finding that all assayed metaphase I spindles had 7 chromosomes also indicates that our method of identifying XXX worms as dumpy individuals is accurate and the high frequency of metaphase II spindles with 6 chromosomes is not a result of misidentifying diploid worms as XXX worms. FISH with an X-specific probe revealed that in 6/6 metaphase II embryos with only 6 DAPI-staining bodies, a single hybridization signal was present in the spindle and 2–3 hybridization signals were present in the polar body (Figure 1D; Supplementary file 3). Because only a single X-hybridization signal was observed in the first polar body in 5/5 spindles from diploids, these results demonstrate that single X univalents are deposited in the first polar body with greater than 50% frequency. X and V univalents are frequently deposited in the first polar body To further investigate the mechanism leading to preferential loss of univalents during meiosis I, we utilized him-8 worms as a more tractable model. It has previously been shown that diakinesis stage him-8 oocytes have 5 autosomal bivalents and two X univalents (Phillips et al., 2005). If segregation of the two X univalents was random, these worms should produce equal numbers of nulloX and diploX ova. Instead, him-8 mutants produce a fivefold higher frequency of nulloX ova over diploX ova (Hodgkin et al., 1979), indicating that both maternal X univalents are lost at some time after diakinesis in a large fraction of embryos. To determine when maternal X univalents are preferentially lost, we imaged both live embryos within him-8 worms expressing GFP::tubulin and mCherry::histone (Figure 2A–E) and also fixed embryos stained with DAPI and anti-tubulin antibodies (Figure 2F–J). We assayed the number of chromosomes (defined here as DAPI-staining or mCherry:histone-positive bodies that would include univalents and bivalents) present at metaphase of meiosis I and II (Figure 2K). At meiosis I metaphase, 7 chromosomes were present in 96% of him-8 embryos (Figure 2B,K), with the remainder having 6 chromosomes. If the two univalents segregated randomly without losing cohesion, 25% of metaphase II spindles would be expected to have 5 autosomes and no X, 50% would have 5 autosomes and 1 X, and 25% would have 5 autosomes and 2 X chromosomes. Instead, 40% of him-8 metaphase II embryos had 5 chromosomes, 55% had 6 chromosomes, and only 5% had 7 chromosomes (Figure 2K). These frequencies differ significantly from those expected from unbiased segregation (chi-square test, two-tailed p < 0.0001), closely match the ratio of nulloX to diploX ova inferred by Hodgkin et al. (1979) and support the hypothesis that the majority of X univalents are eliminated between metaphase I and metaphase II. These maternal chromosome counts are also unaffected by nulloX or diploX sperm that might contribute to phenotype-based progeny counts. Figure 2 with 1 supplement see all Download asset Open asset X univalents are preferentially lost between metaphase I and metaphase II in him-8 mutants. Z projections of living (A–E) and fixed (F–J) C. elegans meiotic embryos viewed down the pole-to-pole spindle axis at metaphase I (A, B, F, G) or metaphase II (C, D, E, H, I, J). mCherry::Histone H2B and GFP::tubulin label the chromosomes and spindle, respectively, in live embryos. Fixed embryos were stained with DAPI, anti-tubulin antibody, and a LacO FISH probe that recognizes a LacO array integrated on the X chromosome. Asterisks indicate polar bodies. Insets show polar bodies that did not fit in the image frame. In (E), ‘s’ denotes a sperm outside of the embryo. Percentages are shown for each outcome (K, L). Bar = 5 μm. https://doi.org/10.7554/eLife.06056.004 To confirm that the two chromosomes lost between these stages were indeed the X univalents, we used FISH with a lac operator probe to detect a multi-copy lac operator array integrated on the X chromosomes in a him-8 background (Figure 2F–J). FISH revealed two X univalents and 7 total DAPI-staining bodies on 93% of all the him-8 metaphase I plates (Figure 2G,L). At metaphase II, FISH revealed at least two X hybridization foci in the first polar body and none on the metaphase plate when the spindle had 5 DAPI-staining bodies (Figure 2H). When 6 DAPI-staining bodies were present on the metaphase II plate, we always observed one X hybridization focus each on the metaphase plate and in the first polar body (Figure 2I). Finally, metaphase II embryos containing 7 DAPI-staining bodies had two X hybridization foci on the metaphase plate and none in the first polar body (Figure 2J). Together, these results demonstrate that both achiasmate X univalents are deposited into the first polar body in 40% of him-8 embryos as compared with the 25% expected from random segregation. To test whether achiasmate autosomes are also placed in the first polar body with higher than random frequency, we analyzed a strain with a lac operator array integrated on chromosome V and bearing a loss of function mutation in the him-8 homolog zim-2, which contributes to chromosome V pairing (Phillips and Dernburg, 2006). Unlike the situation with him-8 and pairing of the X, redundancy between ZIM proteins may contribute to chromosome V pairing. Phillips and Dernburg reported only 72% of diakinesis oocytes with 7 rather than 6 DAPI-staining bodies in a zim-2 mutant, and our zim-2 strain with lacO(V) had only 62% of diakinesis oocytes with 7 DAPI-staining bodies (Figure 2—figure supplement 1F). FISH revealed two distinct chromosome V hybridization foci and 7 DAPI-staining bodies on 41% of metaphase I spindles (Figure 2—figure supplement 1A,E). Starting with 41% achiasmate V's, random segregation should yield 10% of metaphase II embryos with both V's in the first polar body (25% of 41%). Instead, FISH revealed 27% of metaphase II embryos had five DAPI-staining bodies on the metaphase plate and chromosome V hybridization foci only in the first polar body (Figure 2—figure supplement 1B,E). Likewise, random segregation of achiasmate V's should yield 10% metaphase II spindles with 7 DAPI-staining bodies on the metaphase II spindle, two distinct chromosome V hybridization foci on the spindle, and none in the first polar body. Only 5% of this embryo class was observed (Figure 2—figure supplement 1D,E). These frequencies are significantly different than those expected from random segregation (chi-square test, two-tailed p < 0.0002). The discrepancy in the fraction of zim-2 oocytes with 7 DAPI-staining bodies at diakinesis vs metaphase I raises the possibility that chromosomes might be systematically undercounted in zim-2 metaphase I spindles (but not in wild-type, him-8, or XXX metaphase I spindles). If this is the case, the two V univalents must be positioned close together on the spindle because 0/57 zim-2 metaphase I plates with 6 DAPI-staining bodies had two widely spaced lacO(V) FISH foci and the deviation between expected and observed nulloV metaphase II spindles would be even greater. Two results strongly indicate that the same mechanisms acting on univalent X's in him-8 mutants also act on V univalents in the zim-2 mutant. First, the fivefold preponderance of metaphase II spindles with 5 DAPI-staining bodies over those with 7 DAPI-staining bodies is similar to him-8. Second, the presence of lacO FISH signal only in the first polar body of metaphase II embryos with 5 DAPI-staining bodies on the spindle is the same in him-8 and zim-2. Thus, achiasmate autosomes, like achiasmate X chromosomes, are preferentially deposited into the first polar body. Univalents biorient at metaphase I and tend to lag during anaphase I To understand the mechanism by which univalent X chromosomes are preferentially deposited in polar bodies, we examined their orientation and position in the spindle. Antibodies specific for the cohesin subunit, REC-8, label a cruciform on metaphase I bivalents (Figure 3A,B) and a single band on metaphase II chromosomes (Figure 3A,C). The single REC-8 bands on wild-type metaphase II chromosomes and on him-8 metaphase I univalents were both oriented perpendicular to the pole-to-pole axis of the spindle (Figure 3D), indicating that him-8 X univalents biorient at metaphase I. him-8 worms expressing GFP::KNL-2, which labels the C. elegans cup-shaped meiotic kinetochores (Dumont et al., 2010), were also analyzed for biorientation and yielded the same conclusion as analysis by REC-8 antibody (Figure 3—figure supplement 1). We also examined the localization of GFP:AIR-2, the aurora B kinase that is essential for the loss of cohesion at anaphase I and which is loaded between homologs of wild-type bivalents in a chiasma-dependent fashion (Rogers et al., 2002). The fluorescence intensity of GFP::AIR-2 was 2.3 times higher on bivalents than univalents at metaphase I of him-8 embryos (Figure 3—figure supplement 2B,C). AIR-2 is normally re-loaded between sister chromatids at metaphase II. GFP::AIR-2 on metaphase II chromosomes was 1.7 times higher than on him-8 metaphase I univalents (Figure 3—figure supplement 2C), indicating that the reduced amount of AIR-2 on metaphase I univalents was not simply a consequence of the smaller size of a univalent relative to a bivalent. AIR-2 is required for the crossover dependent, prometaphase, partial removal of REC-8 from between homologs in a wild-type bivalent, an event proposed to be essential for loss of cohesion at anaphase I (Severson and Meyer, 2014). Consistent with the low levels of AIR-2, him-8 univalents had 1.7 ± 0.3 times the intensity of REC-8 staining as the inter-homolog region of bivalents in the same spindle (Figure 3B; n = 8 embryos, two-tailed p = 0.04, chi square relative to expected 1.0). Figure 3 with 2 supplements see all Download asset Open asset X univalents biorient at metaphase I in him-8 embryos. (A) Cartoon diagram of REC-8 staining on bivalents and univalents. (B and C) Anti-REC-8 staining of metaphase I and metaphase II embryos with bivalents (yellow arrow head) and univalents (white arrow head). In him-8 embryos, univalents at metaphase I have a single band of REC-8 with the same orientation seen on normal chromosomes at metaphase II. (D) Quantification of the orientation of univalents by offset angle from the metaphase plate, 0° corresponds to perfect biorientation and 90° corresponds to perfect is on the in all Bar = 5 μm. Because X univalents biorient at metaphase I as AIR-2, which is required for loss of cohesion at anaphase I in C. elegans et al., 2002; et al., and twice as REC-8, we that univalents might be toward both spindle poles and lag on the anaphase spindle as to lose To test this we did of him-8 embryos expressing GFP::tubulin and on the of anaphase I. of him-8 embryos at anaphase I had one or two chromosomes = compared to of wild-type embryos = (Figure In of living him-8 embryos with chromosomes at anaphase I, two chromosomes be chromosome as a single either toward the cortex or into the embryo in of embryos = indicating that cohesion between sister chromatids is and that univalents are not broken or during anaphase. At anaphase II, only 10% of him-8 embryos chromosomes = and wild-type embryos had chromosomes, suggesting that chromosomes are by the presence of univalents at meiosis I. Figure Download asset Open asset X univalents lag at anaphase I. (A) images of a living wild-type embryo anaphase I show chromosomes as two distinct (B) images of a living him-8 embryo show a chromosome at anaphase I. (C–E) Z projections of fixed anaphase I embryos. (C) LacO FISH of a wild-type strain with a LacO array integrated on the X chromosome shows normal segregation of two X homologs from one X bivalent. (D) LacO FISH shows that a chromosome in him-8 is the X. (E) LacO FISH of a zim-2 strain with a LacO array integrated on chromosome V showing a univalent V at anaphase I. is to the in all Bar = 5 μm. Figure 5 Download asset Open asset The contractile moves the chromosomes of him-8 embryos. (A) of anaphase I in a him-8 strain with and mCherry::Histone The the chromosomes to in the polar body. (B) of him-8 anaphase I embryos in which a chromosome toward the cortex or into the embryo univalents more frequently toward the cortex during both and anaphase. of the required for polar body eliminated only the anaphase two-tailed p by him-8 vs him-8 = him-8 vs him-8 = him-8 vs him-8 = him-8 vs him-8 = p from Pearson's chi-squared him-8 vs 50% = him-8 vs 50% = him-8 vs 50% = him-8 vs 50% = (C) diagram the position of by the contractile the pole-to-pole spindle axis was images from sequences showing at different the of the spindle. (D) position of contractile the pole-to-pole spindle axis in wild-type embryos and in him-8 embryos with no chromosomes, chromosomes that end up at the cortex or chromosomes that end up in the embryo Bar = 5 μm. To confirm that the chromosomes are X univalents, we used We found that the X-specific FISH probe labeled one or two chromosomes at anaphase I of him-8 embryos, indicating that chromosomes are X univalents (Figure of fixed him-8 anaphase I embryos with chromosomes had two distinct chromosomes had a single no other chromosomes on the spindle indicating that the two X univalents were close to in these embryos. The had one chromosome and one chromosome in one of the chromosome embryo had a chromosome that was a These results suggest that one or both X univalents lag in up to of him-8 embryos. results were for chromosome V in zim-2 40% of metaphase I embryos have univalent V's (Figure 2—figure supplement 1). 27% or over of anaphase I spindles expected to have V had a chromosome. 100% of these chromosomes were chromosome V as assayed by FISH (Figure These results indicate that achiasmate autosomes lag at anaphase I, like achiasmate X chromosomes. The meiotic contractile X univalents in the first polar body that chromosomes were univalents, we if univalents that lagged were to biased segregation at anaphase I. To we of embryos from him-8 worms expressing GFP::tubulin and as well as embryos from him-8 worms expressing these with to label the (Figure analysis revealed that of chromosomes toward the cortex and the polar body of him-8 embryos during anaphase I = (Figure indicating that preferential of univalents into the first polar body contribute to the higher than random frequency of metaphase II spindles with 5 autosomes and no X. Because the polar body contractile toward the of the anaphase spindle it normally we that the preferential of chromosomes toward the cortex might result from of contractile the chromosomes (Figure In of the polar body contractile when homologs have separated by 2.3 and polar body when homologs have separated by et al., The bias of univalents that toward the cortex of contractile not be by by the polar body. we separated univalents into two univalents that to one while the chromosome were separated by than and univalents that to one only after the chromosome