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. 2011 Mar;187(3):685–699. doi: 10.1534/genetics.110.124958

An Asymmetric Chromosome Pair Undergoes Synaptic Adjustment and Crossover Redistribution During Caenorhabditis elegans Meiosis: Implications for Sex Chromosome Evolution

Jonathan V Henzel *,1, Kentaro Nabeshima †, Mara Schvarzstein ‡, B Elizabeth Turner *, Anne M Villeneuve ‡,§, Kenneth J Hillers *,2
PMCID: PMC3063665  PMID: 21212235

Abstract

Heteromorphic sex chromosomes, such as the X/Y pair in mammals, differ in size and DNA sequence yet function as homologs during meiosis; this bivalent asymmetry presents special challenges for meiotic completion. In Caenorhabditis elegans males carrying mnT12, an X;IV fusion chromosome, mnT12 and IV form an asymmetric bivalent: chromosome IV sequences are capable of pairing and synapsis, while the contiguous X portion of mnT12 lacks a homologous pairing partner. Here, we investigate the meiotic behavior of this asymmetric neo-X/Y chromosome pair in C. elegans. Through immunolocalization of the axis component HIM-3, we demonstrate that the unpaired X axis has a distinct, coiled morphology while synapsed axes are linear and extended. By showing that loci at the fusion-proximal end of IV become unpaired while remaining synapsed as pachytene progresses, we directly demonstrate the occurrence of synaptic adjustment in this organism. We further demonstrate that meiotic crossover distribution is markedly altered in males with the asymmetric mnT12/+ bivalent relative to controls, resulting in greatly reduced crossover formation near the X;IV fusion point and elevated crossovers at the distal end of the bivalent. In effect, the distal end of the bivalent acts as a neo-pseudoautosomal region in these males. We discuss implications of these findings for mechanisms that ensure crossover formation during meiosis. Furthermore, we propose that redistribution of crossovers triggered by bivalent asymmetry may be an important driving force in sex chromosome evolution.

MEIOSIS is the specialized cell division through which sexual eukaryotes produce haploid gametes. Several basic features of meiosis are widely conserved among eukaryotes. Following premeiotic DNA replication, proteinaceous structures known as axial elements assemble along the length of each chromosome, and homologous chromosomes pair and align. Following homolog pairing, another structure known as the synaptonemal complex (SC) assembles between paired chromosomes (reviewed in Page and Hawley 2003). In the context of paired and synapsed homologous chromosomes, meiotic crossing over is completed. Crossing over is initiated by the programmed introduction of DNA double-strand breaks during prophase I; repair of a subset of these breaks results in formation of crossovers (reviewed in Keeney 2001). These crossovers, in conjunction with sister-chromatid cohesion, form the basis of physical connections between homologs that persist until the metaphase I to anaphase I transition and allow bi-orientation of homologous chromosomes toward opposite poles of the meiosis I spindle (reviewed in Petronczki et al. 2003). Separation of homologous chromosomes at anaphase I, followed by separation of sister chromatids at anaphase II, produces meiotic products (gametes) with half the chromosome complement of the progenitor cell. Fusion of two gametes recreates the original ploidy in the next generation.

Synapsis of homologous chromosomes is a critical event during meiosis; in most organisms, synaptic failure results in errors in meiotic chromosome segregation. An important first step in synapsis is the pairing of homologous chromosomes. In most eukaryotes, homolog alignment is facilitated by tethering of chromosomes to the nuclear envelope, which promotes establishment and/or maintenance of contacts between homologous chromosomes (at the expense of contacts between nonhomologous chromosomes). This process of pairing is coupled with assembly of the synaptonemal complex, which ensures that, under normal circumstances, synapsis takes place strictly between homologs (for recent reviews, see Ding et al. 2010; Mlynarczyk-Evans and Villeneuve 2010).

In the nematode Caenorhabditis elegans, specialized chromosome regions known as pairing centers or homolog recognition regions play a central role in pairing and synapsis (Rosenbluth and Baillie 1981; McKim et al. 1988; Herman and Kari 1989; Villeneuve 1994; MacQueen et al. 2005). These pairing centers (PCs), which act to stabilize pairing and promote synapsis, are enriched for short repetitive sequence elements (Phillips et al. 2009) that promote binding of one of four related proteins (HIM-8, ZIM-1, -2, and -3) required for pairing-center activity (Phillips et al. 2005; Phillips and Dernburg 2006).

The process of synapsis involves assembly of the SC protein structure between paired chromosomes. SC assembly is important for normal levels of crossovers in many organisms; mutation of SC central region components reduces or eliminates meiotic crossing over in C. elegans, Drosophila, and Saccharomyces cerevisiae (Sym and Roeder 1994; Page and Hawley 2001; MacQueen et al. 2002). Recent work has further illuminated the role of the C. elegans SC in establishment of the crossover landscape: in this organism, the SC acts to locally promote crossover formation while also acting to limit the total number of crossovers per chromosome through an inhibitory role (Hayashi et al. 2010).

Under normal circumstances, establishment of synapsis is closely coupled to pairing of homologous chromosomes. However, homology between chromosomes is not required for their synapsis. This can be seen in some C. elegans mutants in which pairing is impaired and SC forms between nonhomologous chromosomes (e.g., htp-1: Couteau and Zetka 2005; Martinez-Perez and Villeneuve 2005; sun-1: Penkner et al. 2007). In addition, nonhomologous synapsis is seen in crossover-suppressed regions of heterozygous reciprocal translocations (MacQueen et al. 2005). These results indicate that synapsis in C. elegans is not strictly limited to homologous chromosomes and underscore the importance of pairing-center-mediated interactions between chromosomes in promoting SC assembly between homologous chromosomes in C. elegans.

Nonhomologous synapsis has also been demonstrated to occur in situations where synapsis takes place between homologous chromosomes that differ in structure. For example, Moses and Poorman (1981) used electron microscopy to examine synapsis during spermatogenesis in mice heterozygous for a tandem duplication of a portion of chromosome 7. In these animals, the two copies of chromosome 7 had axes of different lengths at the end of zygotene, reflecting the difference in physical length of the two chromosomes. Synapsis early in pachytene was apparently dependent upon homology; one copy of the duplication looped out from the bivalent to form an axial “buckle.” However, as pachytene progressed, local desynapsis was observed in the vicinity of the buckle. Concomitantly, the lengths of the two axes became equalized through shortening of the longer axis. Later in pachytene, full synapsis was reestablished; the “buckle” disappeared, the two chromosome axes were approximately equivalent in length, and in the vicinity of the duplication heterologous sequences were synapsed. This process has been termed “synaptic adjustment” (reviewed in Zickler and Kleckner 1999).

While synaptic adjustment can lead to establishment of end-to-end synapsis between chromosomes with dissimilar lengths, there are many cases of naturally occurring chromosome pairs where synapsis and crossovers are restricted to a limited domain of a chromosome pair—most notably, to heteromorphic sex chromosomes such as the X/Y pair in mammals (Bergero and Charlesworth 2009). In these cases, the two chromosomes can be widely divergent both in size and sequence along most of their lengths, while sharing a short domain of homology (referred to in mammals as the pseudoautosomal region). In such cases, crossover formation is limited to the region of homology. The evolutionary history of asymmetric sex bivalents has been inferred, from both comparisons of sequence divergence between homologous genes and examination of structural organization in phylogeny (reviewed in Wilson and Makova 2009). However, how the functions of meiotic chromosome structures may have contributed to mechanisms of evolution of heteromorphic sex chromosomes has not been widely investigated.

Here, we investigate the meiotic behavior of a historically recent heteromorphic neo-sex chromosome bivalent during meiosis in C. elegans. In males heterozygous for the X;IV fusion chromosome mnT12 and normal IV, a region capable of homologous synapsis (the chromosome IV portion of mnT12) is contiguous with a chromosomal region with no homologous pairing partner (the X chromosome portion of mnT12) (Sigurdson et al. 1986).

We demonstrate that the unpaired X axis has a morphology distinct from that of paired/synapsed autosomal axes. We also demonstrate that a chromatin feature characteristic of the X chromosome portion of mnT12 spreads onto the conjoined autosomal portion of the fusion chromosome, despite the fact that it is engaged in synapsis. We demonstrate the occurrence of synaptic adjustment in C. elegans by showing that synapsis at the left end of IV in mnT12/+ males is homologous in early pachytene, but nonhomologous in late pachytene. Finally, we show that meiotic crossovers along the chromosome IV portion of the mnT12/IV bivalent are redistributed relative to controls, resulting in an increased occurrence of crossovers far from the X;IV fusion point. This crossover redistribution has the effect of generating a neo-pseudoautosomal region between the two chromosomes in the asymmetric bivalent. We discuss the implications of these findings for mechanisms that drive the evolution of sex chromosomes.

MATERIALS AND METHODS

Genetics:

C. elegans strains were cultured at 20° under standard conditions (Brenner 1974). Wild-type strains N2 Bristol and CB4856 were used, along with chromosome fusion strain mnT12 (X; IV) and mutant strain unc-3(e151) (X).

Cytological analyses:

mnT12/+ males:

Immunostaining of dissected gonads from 24-hr post-L4 adult males was performed as in Nabeshima et al. (2004) with some modifications. Briefly, males were dissected on microscope slides and freeze-cracked into ice-cold methanol for 1 min. Samples were then fixed in 3.7% formaldehyde in 1× PBS + 0.08 m EDTA + 1.6 mm MgSO4 for 30 min at room temperature. The following primary antibodies were used at the indicated dilutions: rabbit α-HIM-3 (1:200). Rabbit α-histone H3 dimethyl-Lys9 (H3K9me2) (Upstate Biotechnology) (1:200). Guinea pig α-SYP-1 (1:100). Experiments involving immunostaining for both H3K9me2 and HIM-3 were performed by first immunostaining with rabbit α-histone H3K9me2 (1:200), followed by washes and incubation with a FITC-labeled α-rabbit F(ab) fragment (1:100). Samples were then washed and incubated with rabbit α-HIM-3 (1:200) and Cy3 α-rabbit (Jackson Immunochemicals) (1:200). Images were obtained as stacks of optical sections acquired at 0.2-μm intervals using the Deltavision deconvolution microscopy system.

N2 males:

Immunostaining of dissected gonads from N2 males was as described (Gonczy et al. 1999; Oegema et al. 2001) with minor modifications. Gonads from young adult males were dissected, fixed, and permeabilized by freeze-cracking in liquid nitrogen followed by soaking in methanol at −20° (Gonczy et al. 1999) for 30 min. Embryos and gonads were rehydrated in PBS, blocked in AbDil (PBS plus 2% BSA, 0.1% Triton X-100) as described by Oegema et al. (2001), and incubated at 4° overnight in 2 μg/ml of mouse α-histone H3K9me2 antibody (Abcam) and rabbit α-HIM-3 (1:200 dilution) (Zetka et al. 1999). After washing with PBST (PBS plus 0.1% Triton X-100), gonads were incubated with PBST containing 1 mg/ml Hoechst 33258 (Sigma) and mounted in 0.5% p-phenylenediamine, 20 mm Tris–Cl, pH 8.8, and 90% glycerol (Oegema et al. 2001). Images were obtained as stacks of optical sections acquired at 0.2-μm intervals using the Deltavision deconvolution microscopy system.

Quantitation of pairing and synapsis of chromosome IV sequences:

FISH combined with immunostaining of dissected gonads from 24-hr post-L4 adult males was performed as in Nabeshima et al. (2004) with some modifications. Briefly, males were dissected on microscope slides and incubated with egg buffer (Edgar 1995) containing 10% Tween-20 for 5 min at room temperature, followed by fixation in egg buffer containing 1% paraformaldehyde for 1 min. After fixation, slides were freeze-cracked into ice-cold 95% ethanol for 5 min. Immunostaining was done with guinea pig α-SYP-1 (1:200) antibody and Alexa 555 α-guinea pig (Invitrogen) (1:400). After immunostaining, slides were post-fixed in PBS containing 4% paraformaldehyde for 10 min before hybridization with FISH probes. Hybridization was done at 37° overnight after heat denaturing at 80° for 10 min followed by stepwise cooling to 37° in 4 min on the OmniSlide flat bed thermal cycler (Thermo). FISH probes were made by labeling the following YACs using the ULYSIS DNA labeling kit (Invitrogen). The probe used for IV-L was a cocktail of Y41H10 and Y59E4 (covering the terminal 600 kb of the left end) labeled with Alexa 488 and for IV-R a cocktail of Y51H4, Y43D4, and Y116A8 (covering the terminal 700 kb of the right end) labeled with Alexa 647. Images were obtained as stacks of optical sections acquired at 0.2-μm intervals using the Deltavision deconvolution microscopy system.

For three full gonads from N2 males and three full gonads from mnT12/+ males, four-color stacks of images were collected with DAPI, IV-L FISH, IV-R FISH, and anti-SYP-1 immunostaining. For each gonad, the transition zone (TZ) and condensation zone (CZ) were identified (on the basis of nuclear morphology). For scorable nuclei between the TZ and CZ, the center of intensity of each FISH focus was determined, and distances were determined between each of the four foci (IV-L to IV-L, IV-R to IV-R, plus four different IV-L to IV-R distances). In cases where two FISH foci were not resolved, the distance measured represented the breadth of the region of maximum staining intensity (typically, 0.2 μm). For each gonad, the region of scored nuclei was divided into five bins of roughly equal size (bins 1–4 are equal in size; bin 5 is larger, due to the binning algorithm used).

For a subset of nuclei, we also measured the distance between each FISH focus and the nearest contiguous stretch of SYP-1 staining. FISH-SYP-1 distances were determined for early pachytene nuclei (bin 1) and for mid/late-pachytene nuclei (corresponding to the seminal vesicle proximal half of bin 3 and the distal half of bin 4). Examination of FISH-SYP-1 distances in control images revealed that ∼90% of FISH foci were within 0.65 μm of a SYP-1 stretch in early pachytene (when all sequences should be synapsed); thus, we operationally define FISH foci within 0.65 μm of a SYP-1 stretch as being associated with SYP-1.

Snip-SNP analysis:

Meiotic crossing over was assayed in control and mnT12/+ animals using SNP markers, as in Nabeshima et al. (2004) and Hillers and Villeneuve (2009). Markers and primers used are listed in Table 1. CB4856 males were mated to mnT12 (experimental) or N2 (control) hermaphrodites, and outcross males were picked. These represent the animals in which crossing over was analyzed. Individual mnT12/CB4856 or N2/CB4856 males were plated with two to three unc-3(e151) (N2 background) hermaphrodites. After 24 hr, males were removed. Mated hermaphrodites were moved to fresh plates every 24 hr. Outcross progeny (non-Unc hermaphrodites and unc-3 males) were picked individually to PCR tubes containing 10 μl 10 mm Tris, pH 8.0, and stored at −80°. Snip-SNP markers were analyzed as in Hillers and Villeneuve (2009).

TABLE 1.

Chromosome IV SNP alleles and corresponding primers

Digested product size (bp)b
SNP Chromosomal position (bp)a Primer sequence Enzymeb N2 CB4856
Y38C1A[2] 151,888 5′-aaataacaggcacctaccgc-3′ XbaI 882 481, 401
5′-ctttgaaggaggactaacgg-3′
pkP4053 1,954,096 5′-acatttagtcacgcgtaggg-3′ HpaII 191, 137, 22 328, 22
5′-gcccgaatctagcacataag-3′
pkP4032 3,494,570 5′-tgtctaccgtatacctggac-3′ RsaI 163, 131 294
5′-atccagctcaaaagtgtgcg-3′
pkP4073 5,513,221 5′-aatacagcagtcgttccgttc-3′ DraI 288, 144 432
5′-tgaacttcatgaaccagcttg-3′
pkP4039 8,397,257 5′-acgaaaaatcacagagcggg-3′ EcoRI 650, 202 852
5′-aatcaacaacggacgacgag-3′
pkP4088 12,975,669 5′-gattatttcagaggagcagagc-3′ HindIII 420 245, 175
5′-catagcacgtggaataaccac-3′
pkP4102 17,392,780 5′-tgcttaaagtcatcgtgtccac-3′ EarI 235, 174 409
5′-tgtaaaccgtatcgaatccgac-3′
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a

Position of the SNP in the N2 chromosome IV sequence (total length of IV: 17,493,785 bp).

b

For each SNP allele, digestion of the amplified product using the designated restriction enzyme yielded products of the specified size.

RESULTS

Experimental system:

A schematic of the karyotypes analyzed in this work is presented in Figure 1. Whereas C. elegans sex determination is usually XX/XO, the fusion chromosome mnT12 (an end-to-end fusion of X and IV) creates a situation in which there are two heteromorphic sex chromosomes. Worms homozygous for mnT12 are hermaphrodites; in this situation, the two copies of mnT12 behave as a typical chromosome pair, able to align and synapse fully along their lengths with a single crossover per meiosis (Hillers and Villeneuve 2003; Martinez-Perez et al. 2008). In contrast, worms heterozygous for mnT12 and normal IV are male (they have a single copy of the X chromosome, provided by mnT12). In these animals, mnT12 and normal IV constitute a highly asymmetric chromosome pair; the chromosome IV portion of mnT12, which is capable of pairing and synapsis with the normal IV, is contiguous with a normally partnerless X.

Figure 1.—

Figure 1.—

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Wild-type and mnT12 karyotypes. Diagrammatic representations of unfused chromosomes X and IV and X;IV fusion chromosome mnT12 in males and hermaphrodites. Horizontally lined areas represent regions of pairing-center activity.

Here, we examine the behavior of this highly asymmetric chromosome pair with respect to several meiotic features: (1) chromosome structure, (2) crossover distribution, and (3) status of alignment and synapsis.

Structural organization of the mnT12/+ bivalent:

We used immunofluorescence analysis to evaluate the status of meiosis-specific structural elements in pachytene nuclei in mnT12/+ males. Specifically, we assessed localization and organization of HIM-3, a major component of the meiotic chromosome axis in C. elegans (Zetka et al. 1999), and SYP-1, a major component of the central region of the synaptonemal complex in C. elegans (MacQueen et al. 2002).

The mnT12/+ bivalent was identified in these experiments using an antibody against histone H3 dimethyl-Lys9 (H3K9me2), a histone modification that is strongly enriched on the male X chromosome (Kelly et al. 2002). Several features of the data are notable.

First, we found that the mnT12-containing bivalent harbors characteristics of both its autosomal and X chromosome components (Figure 2, A and B). One portion of the mnT12-containing bivalent stains strongly with anti-H3K9me2; this portion of the bivalent loads HIM-3 but not SYP-1. This is comparable to the behavior of the single X in wild-type males (Jaramillo-Lambert and Engebrecht 2010). The remaining portion of the mnT12 bivalent loads SYP-1 and HIM-3, as is normal for an autosome pair.

Figure 2.—

Figure 2.—

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Pachytene chromosome structures in wild-type and mnT12/+ males. (A and B) Immunolocalization of histone H3 dimethyl-Lys 9 (H3K9me2) and axis component HIM-3 (A) or SC central region protein SYP-1 (B) in mnT12/+ males. Images are partial projections of single nuclei at the mid-pachytene stage. One portion of the mnT12 bivalent stains strongly with anti-H3K9me2; this portion loads HIM-3 (A) but not SYP-1 (B); the HIM-3 in this region has a convoluted appearance. H3K9me2 staining spreads into the conjoined synapsed region, but more weakly and along one side. (C) Immunolocalization of HIM-3 and H3K9me2 in N2 males. Images are partial projections of individual nuclei at mid-pachytene. The single X stains strongly with anti-H3K9me2; the HIM-3 on this chromosome has a convoluted appearance. Scale bars, 2 μm.

Second, we noted that a portion of the mnT12 axis has a convoluted appearance. Specifically, the morphology of the unpaired chromosome axis in mnT12/+ males differs from that of the synapsed autosomes. In Figure 2A, HIM-3 staining can be seen along the full length of mnT12; however, one segment of the HIM-3 staining appears to be in a helical configuration, while the remainder assumes an extended linear configuration (Figure 2A, Left). Similar results are seen when HIM-3 staining patterns are examined in wild-type males (Figure 2C). Thus, the X chromosome axis that assembles in males is morphologically distinct from that of the synapsed autosomes.

Finally, we also noted that histone H3 dimethyl-Lys9 staining extended from the asynapsed portion of mnT12 onto the synapsed portion, albeit more weakly and predominately along only one of the two homologs. For example, Figure 2B shows images from a colocalization experiment in mnT12/+ males involving SYP-1 and H3K9me2, in which one chromosome region stains strongly for H3K9me2 and lacks SYP-1, while weaker H3K9me2 staining can be seen to extend the length of the adjacent SYP-1-staining portion of the fusion chromosome (corresponding to the region engaged in synapsis), but along only one side (Figure 2B). A similar effect can be seen when colocalization of HIM-3 and H3K9me2 is examined (Figure 2A). This is in contrast to the situation in wild-type males, where strong H3K9me2 staining is limited to the single X chromosome in early pachytene (Figure 2C; Kelly et al. 2002). This spreading of H3K9me2 staining suggests that the heterochromatic nature of the single male X is capable of spreading onto adjacent chromosome regions engaged in synapsis and is not solely limited to unpaired regions.

Distribution of crossovers along chromosome IV is altered in mnT12/+ males:

To evaluate the effect of bivalent asymmetry on meiotic recombination, we assayed the frequency and distribution of meiotic crossovers along the length of chromosome IV in wild-type males with normal karyotype and in males heterozygous for mnT12 (Figure 3; Table 2; Table 3).

Figure 3.—

Figure 3.—

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Crossing over along chromosome IV in control and mnT12/+ males. (A) Schematic of the physical locations of the SNP markers used in crossover mapping experiments (to scale). For specific details, see Table 1. (B) The proportion of all chromosome IV crossovers occurring in a given interval for both control (gray bars) and mnT12/+ (black bars) males. (C) Genetic maps of the assayed portion of IV in control (Top) and mnT12/+ (Bottom) males. Map length and marker positions are to scale; numbered intervals correspond to those in A. Corresponding markers are connected by dotted lines. Red intervals: the ratio of the interval size in mnT12/+ to control males is <0.5. Green intervals: the mnT12/+ to control ratio is >2.0. (D) For control and mnT12/+ males, the positions of the two exchanges in DCO meiotic products are indicated, as well as the number (n) of each class that was detected. For those pairs of intervals in which there were five or more expected DCOs, the coefficient of coincidence (“C”) and P-value from Fisher's test for independent occurrence of coincident crossovers are provided (see Evidence for crossover interference).

TABLE 2.

Classes of chromosome IV meiotic products

No crossover (%)a Single crossover (%)a Double crossover (%)a n Map length (cM)
N2/CB4856 120 (44.3) 139 (51.3) 12 (4.4) 271 60.1
mnT12/CB4856 128 (47.9) 132 (49.4) 7 (2.6) 267 54.7
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a

The proportion of meiotic products in each of these three classes was not significantly different between experimental and control (P = 0.42; χ2).

TABLE 3.

Distribution of crossovers along the length of chromosome IV

Crossovers in interval (%)a
1 2 3 4 5 6
N2/CB4856 68 (25.1) 30 (11.1) 13 (4.8) 9 (3.3) 14 (5.2) 29 (10.7)
mnT12/CB4856 14 (5.2) 7 (2.6) 4 (1.5) 16 (6.0) 27 (10.1) 78 (29.2)
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Numbers in parentheses indicate recombination frequency.

a

The distribution of crossovers along IV differs significantly between experimental and control data sets: P < 0.0001 (contingency χ2).

To detect crossovers, we generated males heterozygous for chromosomes derived from the Bristol N2 strain and the Hawaiian isolate CB4856. mnT12/CB4856 heterozygous males have one copy of mnT12 (derived from Bristol N2) and one copy of CB4856-derived IV. N2/CB4856 males have a single X and are heterozygous for N2- and CB4856-derived IV. In these animals, mapped SNP markers that differ in these two strain backgrounds provide a dense array of possible genetic markers (Hillers and Villeneuve 2009 and references therein). mnT12/CB4856 males (experimental) or N2/CB4856 males (control) were mated to N2 hermaphrodites homozygous for unc-3(e151). Outcross (non-Unc) progeny will all inherit an N2-derived chromosome IV from the hermaphrodite parent. The chromosome IV sequences contributed by the male parent can be determined by genotyping SNP markers; the position of any crossover that occurs can be detected through the content of N2- and CB4856-derived SNP alleles. We assayed segregation of seven SNP alleles, spanning 98.5% of the physical length of wild-type IV (Table 1; Figure 3A). This allows detection of the vast majority of crossovers and also allows detection of multiply exchanged chromosomes.

The total amount of crossing over on IV in mnT12/CB males was comparable to that seen in controls. In mnT12/CB males, the assayed region of chromosome IV had a map length of 54.7 cM, a value not significantly different from that seen in control males (60.1 cM) (Table 2). Double-crossover chromatids represented 2.6% of all chromatids assayed, which is also not significantly different from controls (4.4%). These results indicate that the asymmetric bivalent formed in mnT12/CB males does not significantly impact the overall number of crossovers formed during meiosis.

The overall distribution of crossovers in mnT12/CB males differs markedly from that seen in the control males. In control males, the distribution of crossovers along chromosome IV shows a strong skewing toward the left end of IV, which is the end containing pairing-center activity. This distribution of crossovers is comparable to that seen in hermaphrodites, indicating that control of the distribution of crossovers along wild-type IV is largely sex-independent (Barnes et al. 1995; Rockman and Kruglyak 2009). In mnT12/CB males, however, crossing over was increased in the right portion of IV (distal to the X;IV fusion point) and decreased in the left portion (close to the fusion point) (Table 3; Figure 3, B and C). Interval 1, at the left end of IV, comprises 10% of the physical length of the chromosome. In control males, 42% of all crossovers occurred in interval 1; in mnT12/CB males, on the other hand, only 9.6% of crossovers occurred in this interval. Conversely, 18% of control crossovers occurred in interval 6, while 53% of experimental crossovers occurred in the same interval. The difference in crossover distribution was highly significant (P < 0.0001; contingency χ2). This suggests that some feature of the asymmetrical bivalent formed in mnT12/CB males has a discouraging effect on crossing over near the IV/X fusion point.

Evidence for crossover interference:

Despite the occurrence of double crossovers, there is still evidence for crossover interference in this data set. We tested for interference for those pairs of intervals for which the numbers of crossovers were sufficient to generate an expected number of double crossovers (DCOs) (DCOexp, based on independent occurrence of crossovers in the two intervals) of five or more. In both control and mnT12/CB data, we found evidence for interference between the two adjacent intervals in the region of the chromosome harboring the most crossovers (intervals 1 and 2 for control; intervals 5 and 6 for mnT12/CB). For intervals 1 and 2 in the control data set, we can calculate a coefficient of coincidence (DCOobs/DCOexp; “C” in Figure 3D) value of 0.13. Moreover, we can also evaluate the likelihood that crossovers occur independently in the two intervals through use of Fisher's exact test for independence. For control intervals 1 and 2, the calculated P-value from Fisher's exact test is 0.0016, providing strong statistical evidence that crossovers do not occur independently in intervals 1 and 2. Likewise, for intervals 5 and 6 in mnT12/CB males, C = 0.25 and P = 0.0069, indicating that crossovers are not occurring independently in intervals 5 and 6 (Figure 3D). Together, these data indicate that, in males, crossovers in adjacent intervals are discouraged.

In contrast, we calculate a C value of 0.55 for the widely spaced intervals 1 and 6 in wild type; Fisher's exact test for independence returns a P-value of 0.17 for this pair of intervals (Figure 3D). This supports the idea that the strength of interference is diminished by distance.

Simultaneous analysis of homolog pairing and synapsis reveals synaptic adjustment in mnT12/+ males:

The results of our recombination analyses revealed that the distribution of crossovers in mnT12/+ males is different from that seen in control males; specifically, the crossover distribution is shifted toward the right end of chromosome IV, such that most crossovers occur far from the X/IV fusion point. To address the possibility that this shift resulted from impaired pairing and/or synapsis at the left end of chromosome IV in mnT12/+ males, we carried out an analysis of pairing of chromosome IV loci in mnT12/+ males and N2 male controls. We analyzed pairing through two-color FISH using probes that recognize the left and right ends of IV (Figure 4A).

Figure 4.—

Figure 4.—

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Pairing of chromosome IV sequences in control and mnT12/+ males. (A) Locations of FISH probes used in these experiments. Green: IV-L probe. Red: IV-R probe. (B) Schematic of male gonad structure. Nuclei in the pachytene zone were divided into five bins of roughly equal size. (C) Cumulative distribution graphs plotting data from two-probe FISH experiments monitoring pairing at opposite ends of IV. Bins 1–5 reflect position of nuclei within the pachytene region, with bin 1 nuclei in early pachytene and bin 5 nuclei in late pachytene or diplotene. In these plots, the distances between each pair of homologous signals (y-axis) are plotted against the percentage of measurements that were equal to or less than that distance (x-axis). In early pachytene (bin 1), similar distributions of interfocus distances are seen in all four data sets. As meiotic prophase progresses, IV-L interfocus distances in mnT12/+ males become larger; a comparable increase is not seen in any of the remaining data sets. Light-green circles: IV-L, N2. Light-red circles: IV-R, N2. Dark-green triangles: IV-L, mnT12/+. Dark-red triangles: IV-R, mnT12/+.

This analysis took advantage of the fact that, in the C. elegans gonad, nuclei progress through meiosis in a spatiotemporal gradient as they travel down the gonad; thus, fixed gonads contain nuclei in various stages of meiosis (Figure 4B). Nuclei in the transition zone are in early stages of meiotic prophase (leptotene, zygotene) and are readily recognized by a characteristic asymmetrical distribution of chromatin within the nucleus. Upon exit from the transition zone, nuclear morphology becomes more symmetrical and individual synapsed chromosomes become readily visible; nuclei in this region are in pachytene. Upon exit from pachytene, nuclei transition through diplotene and enter the condensation zone; chromosomes condense, and nuclear DNA becomes confined to a single compact domain (Shakes et al. 2009). In the experiments described here, we assayed pairing in all scorable nuclei between the end of the transition zone and the beginning of the condensation zone; the vast majority of these nuclei will have been in pachytene, with a few in diplotene. For each nucleus scored, the distance between the two IV-L FISH foci was determined, as was the distance between the two IV-R foci. As a control, distances between IV-L and IV-R foci were also determined.

For an initial analysis of pairing in these animals, we determined the median distances between IV-L sequences and between IV-R sequences for the full set of nuclei scored for each genotype. The median interfocus distances in N2 males reflect the fact that chromosome IV was paired throughout pachytene (IV-L: median distance = 0.43 μm; IV-R: median distance = 0.47 μm; n = 283). In mnT12/+ males, however, the median IV-L interfocus distance was >0.7 μm (IV-L: median distance = 0.74 μm; n = 244). This is not a chromosome-wide effect: the median IV-R distance, 0.49 μm, was not significantly different from that seen in N2 males (P = 0.1061; Mann–Whitney). These results indicate that pairing of chromosome IV is impaired specifically at the left end in mnT12/+ males. This is an unexpected result, as the left end of chromosome IV contains the pairing center, a region that plays an integral role in establishment of pairing and synapsis (MacQueen et al. 2005). Thus, we performed a time-course analysis to investigate the pairing state of chromosome IV in wild-type and mnT12/+ males in more detail. To do so, we divided the scored nuclei in each imaged gonad into five bins of roughly equal size (Figure 4B). This analysis accommodates gonads of different size and allows comparison of pairing kinetics among samples.

Early in pachytene, there was no apparent difference in chromosome IV pairing when mnT12/+ males were compared to wild type (Table 4). The median bin 1 IV-L distance in mnT12/+ males was not significantly different from the median bin 1 IV-L distance observed in N2 males (P = 0.1071; Mann–Whitney). However, IV-L sequences in mnT12/+ males become unpaired as prophase progresses; in bins 2–5, mnT12/+ IV-L distances differ significantly from IV-L distances in the corresponding control bin (P < 0.0001 in each case). This dramatic increase in interfocus distance was not seen for chromosome IV loci in N2 males, nor was it seen when pairing of IV-R was examined in mnT12/+ males (Figure 4C). These results indicate that initial pairing of IV-L is successfully established in mnT12/+ males, but is subsequently disrupted or lost.

TABLE 4.

Pairing of chromosome IV sequences: temporal analysis

Strain Bin n Median IV-L distance (μm) Median IV-R distance (μm)
Control 1 54 0.44 0.42
2 54 0.42 0.47
3 54 0.42 0.51
4 54 0.43 0.51
5 67 0.46 0.51
mnT12/+ 1 46 0.47 0.46*
2 46 0.67** 0.47
3 46 0.83** 0.52
4 46 0.87** 0.47
5 60 0.81** 0.54
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*, ** Significantly different from the corresponding control data set. *P = 0.025. **P < 0.0001 (Mann–Whitney).

To determine if the defective pairing seen at IV-L in mnT12/+ males correlated with synaptic defects, we examined synapsis in N2 and mnT12/+ males by examining SYP-1-staining patterns through indirect immunofluorescence. Qualitatively, no obvious differences in SYP-1 loading patterns were seen in mnT12/+ males; thus, there does not seem to be a gross defect in synapsis in these animals (data not shown). To examine the synapsis state of chromosome IV loci, we looked at the association of SYP-1 staining and FISH signals for the left and right ends of IV. In particular, we focused on two regions of each gonad: early pachytene (corresponding to bin 1, above) and mid/late pachytene (corresponding to parts of bins 3 and 4). Within the relevant regions of each imaged gonad, we determined the distance from each FISH focus to the nearest stretch of SYP-1 staining. A FISH signal was defined as being SYP-1 associated if the distance from the center of the FISH signal to the center of the nearest SYP-1 stretch was ≤0.65 μm (see materials and methods).

Synapsis of chromosome IV sequences in early pachytene was efficient in both N2 and mnT12/+ males (Figure 5A). In N2 males, both IV-L signals were associated with SYP-1 staining in every early pachytene nucleus examined (n = 54); the same was true for IV-R. In mnT12/+ males, early pachytene synapsis was nearly as efficient: both IV-L FISH signals were SYP-1 associated in 94% (n = 48) of nuclei, and both IV-R FISH signals were SYP-1 associated in 98% (n = 48) of nuclei. These results indicate that homologous synapsis of chromosome IV sequences is successfully established in both N2 and mnT12/+ males in early pachytene.

Figure 5.—

Figure 5.—

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Synapsis of chromosome IV sequences in control and mnT12/+ males. (A) Stacked bar graph showing the proportion of nuclei in which both, one, or neither FISH signals were within 0.65 μm of a stretch of SYP-1 (defined as SYP-1 associated; see materials and methods) for early and mid/late-pachytene nuclei from control and mnT12/+ males. In all cases, >85% of nuclei assayed had both FISH signals associated with SYP-1 staining. (B) Cumulative distribution graphs plotting the distances between each pair of homologous signals (y-axis) against the percentage of measurements that were equal to or less than that distance (x-axis) for the subset of nuclei in which both FISH signals were associated with a SYP-1 stretch. Green symbols correspond to IV-L distances, while red signals correspond to IV-R. Dark-green and dark-red triangles represent data from mnT12/+ males, while light-green and dark-red circles represent data from N2 males. (C) Combined SYP-1 immunofluorescence and chromosome IV FISH in mnT12/+ male nuclei from early pachytene (Left) and mid/late pachytene (Right). Images shown are partial projections of nuclei highlighting relationships between FISH signals and SYP-1 staining. In early pachytene, most IV-L (green) and IV-R (red) FISH signals are paired and associated with the same SYP-1 stretch. In mid/late pachytene, IV-R FISH signals remain paired, while IV-L FISH signals are commonly separated but associated with the same stretch of SYP-1. Scale bar, 2 μm. (D) Schematic of mnT12/IV synapsis in early pachytene (left) and late pachytene (right). Green and red stars correspond to IV-L and IV-R FISH signals, respectively. The displacement of IV-L FISH signals in late pachytene reflects the heterologous synapsis caused by synaptic adjustment.

In mid/late pachytene, chromosome IV sequences remained synapsed in the vast majority of cases. In N2 males, both IV-L signals were SYP-1 associated in 100% (n = 52) of nuclei examined, while both IV-R signals were SYP-1 associated in 90% (n = 52) of nuclei. In mnT12/+ males, both IV-L signals were SYP-1 associated in 85% (n = 47) of nuclei, while both IV-R signals were associated with SYP-1 staining in 100% (n = 47) of nuclei. This indicates that the failure to maintain pairing of IV-L sequences in mnT12/+ males is not due to a loss of synapsis but instead results from some alteration in the nature of synapsis at IV-L in mnT12/+ males.

To investigate this further, we examined the distance between FISH foci specifically in nuclei in which both foci were associated with SYP-1 staining. The data are presented as cumulative distribution graphs in Figure 5B. In early pachytene nuclei, there were no significant differences in the distribution of interfocus distances among the four data sets. In mid/late-pachytene nuclei, however, the distribution of IV-L distances in mnT12/+ males differed significantly (P < 0.0001, Mann–Whitney) from the distribution of IV-L distances in N2 males; a corresponding difference was not seen at IV-R. Thus, in mnT12/+ males, homologous synapsis of IV-L is established in early pachytene; in mid/late pachytene, IV-L sequences remain SYP-1 associated (and thus synapsed), but are unpaired. This suggests that IV-L synapsis in mid/late pachytene in mnT12/+ males is frequently nonhomologous. Inspection of individual mid/late-pachytene nuclei in mnT12/+ males confirms this: IV-L FISH signals were commonly separated and unpaired, but associated with the same stretch of SYP-1 staining (Figure 5C); similar nuclei were not seen in N2 males.

Taken together, our results indicate that chromosome IV sequences in mnT12/+ males achieve normal homologous synapsis in early pachytene. As pachytene progresses, however, the homologous nature of the IV-L associations is lost; IV-L sequences are still typically SYP-1 associated, but are commonly displaced along the same stretch of SYP-1. This indicates that synaptic adjustment at the left end of chromosome IV is taking place in mnT12/+ males (Figure 5D).

DISCUSSION

Pachytene organization of the X chromosome in C. elegans males:

Our cytological analysis of wild type and mnT12/+ males has revealed structural features of the X chromosome not previously described. We find that the single male X chromosome axis appears convoluted during pachytene, while synapsed axes have an extended linear configuration. In optimal nuclei, the X axis appears to have a helical structure (Figure 2). This may reflect the underlying state of chromosome axes during pachytene. A similar configuration of unpaired axes has been seen in diplotene nuclei as SC disassembly takes place (Nabeshima et al. 2005). The observed structural differences between paired and unpaired axes suggest that the process of synapsis is associated with straightening of intrinsically coiled axes. This could have the effect of placing synapsed axes under mechanical stress. Cycles of establishment and release of stress along chromosome axes have been proposed to play important roles in a variety of processes during meiosis, including crossover control (Kleckner 1996; Zickler and Kleckner 1999). However, our data do not distinguish whether axis straightening is a prerequisite for, or a consequence of, synapsis.

Similar to the single X in wild-type males, the majority of the length of the X chromosome component of mnT12 does not load the central region component SYP-1, despite being contiguous with synapsed chromosome IV sequences (Figure 2). This indicates that, under normal circumstances, SYP-1 loading requires paired axes; SYP-1 protein does not spread onto the adjacent unpaired axis even if loading has been previously nucleated in a region of paired axes.

Synaptic adjustment in C. elegans:

In C. elegans, pairing and synapsis of homologous chromosomes is accomplished through a process involving chromosome regions known as pairing centers or homolog recognition regions; under normal circumstances, synapsis in C. elegans occurs between homologous sequences. However, homology is not a prerequisite for synapsis; in animals heterozygous for a reciprocal translocation, pairing centers can drive synapsis of nonhomologous regions (MacQueen et al. 2005). The same study also found evidence for a mechanism in C. elegans that acts to equalize the lengths of synapsed axes of chromosomes of different length. A similar phenomenon has been observed in other organisms and is commonly referred to as synaptic adjustment (Moses and Poorman 1981).

Early studies of synaptic adjustment suggested that there were two phases of synapsis in mammals. The initial synapsis was thought to be dependent upon homology. Any differences in axial length were then accommodated through the process of synaptic adjustment, presumably involving local desynapsis, equalization of axial lengths, and subsequent heterosynapsis (Moses and Poorman 1981). Our results suggest that a similar progression of events takes place during meiosis in mnT12/+ males. Initial synapsis is directed by the chromosome IV pairing center and occurs between homologous sequences (as indicated by our FISH results; Table 4; Figure 4). However, the axes of the X; IV fusion chromosome and chromosome IV are different in length; as pachytene proceeds, synaptic adjustment takes place, resulting in heterosynapsis at the L end of chromosome IV (Figure 5).

Our data suggest that synaptic adjustment in mnT12/+ males does not result in end-to-end synapsis of mnT12 and IV. In gonads from mnT12/+ males stained with antibodies recognizing H3K9me2 and SYP-1, late-pachytene nuclei commonly have a chromosomal region that stains strongly with H3K9me2 (corresponding to the X chromosome portion of mnT12), which does not colocalize with SYP-1 staining (data not shown). This suggests that synaptic adjustment in these males does not result in complete end-to-end synapsis of IV and mnT12 and further implies that there are limits to the ability of synaptic adjustment to equalize axial lengths.

It is interesting to note that the end of chromosome IV that engages in heterosynapsis in mnT12/+ males is the end that contains the PC. This unexpected result suggests two nonexclusive possibilities. First, the pairing–promoting activity of the PC and associated proteins may be active only (or predominantly) during early meiotic prophase and then diminish as pachytene progresses. Several lines of evidence suggest that this is indeed the case:

  1. PC-dependent patches of the nuclear envelope components SUN-1 and ZYG-12, which correspond to sites of attachment of chromosomes to the cytoskeletal motility apparatus, form during the transition zone (corresponding to leptotene/zygotene) and then later disperse after synapsis is completed (Penkner et al. 2007, 2009; Sato et al. 2009). This suggests that the chromosome mobility- and association-promoting activities of PCs may peak during early meiotic prophase, prior to pachytene. Consistent with this, the ZIM proteins required for autosomal PC function form chromosome-associated foci that peak in intensity in transition zone and early pachytene nuclei. These foci decrease in intensity as pachytene proceeds (Phillips and Dernburg 2006).

  2. PC-mediated synapsis-independent stabilization of homolog pairing reaches peak levels early in the pachytene region of the germline and then fades as prophase progresses (MacQueen et al. 2002). The idea that pairing centers lose preferential association as prophase progresses is further supported by the fact that PC ends of chromosome do not remain preferentially associated as chromosomes desynapse in diplotene (Albertson et al. 1997).

Second, the forces that drive synaptic adjustment may become stronger as pachytene progresses. Thus, early in prophase, pairing-center activity drives homosynapsis of IV-L in mnT12/+ males. Later in pachytene, synaptic adjustment forces are stronger than the forces mediating associations between pairing centers, leading to heterosynapsis.

Bivalent asymmetry and accumulation of chromatin modification:

The single X chromosome in C. elegans males is largely transcriptionally quiescent during meiosis and accumulates high levels of histone H3 lysine 9 dimethylation (H3K9me2) (Reinke et al. 2000; Kelly et al. 2002). Furthermore, H3K9me2 also accumulates preferentially on unsynapsed chromosomal regions in certain meiotic mutants (Bean et al. 2004; K. Nabeshima, unpublished results), supporting the hypothesis that chromatin is modified in response to a lack of pairing and/or synapsis. A similar phenomenon is observed in mammals: unsynapsed regions accumulate specific chromatin modifications, and even heterosynapsis can prevent accumulation of such modifications (Mahadevaiah et al. 2001; Baarends et al. 2005; Turner et al. 2006). Here, we find that, in mnT12/+ males, histone H3 lysine 9 dimethylation is found not only on the single unpaired X but also on the conjoined chromosome IV, which has a pairing partner. Moreover, we see evidence for H3K9me2 staining in regions that are engaged in synapsis, as evidenced by colocalization of H3K9me2 and SYP-1. These results reveal that synapsis does not necessarily prevent H3K9me2 accumulation in C. elegans germ cells. A similar result has been seen in female chickens, in which heterosynapsed Z and W chromosomes also accumulate H3K9me2 (Schoenmakers et al. 2009).

Our results suggest that, once nucleated, the H3K9me2 accumulation associated with the single X chromosome can spread onto adjacent chromosome IV sequences that would not be targeted in wild-type males. These results are similar, and possibly analogous, to those of Ercan et al. (2009), who showed, using chromatin immunoprecipitation, that the C. elegans dosage compensation complex can spread from the X chromosome (in this case in hermaphrodites) onto conjoined autosomal sequences that do not load dosage compensation proteins under normal circumstances (albeit in this case, spreading was limited to the first few megabases of autosome sequences). Taken together, these results provide support for models in which regulatory chromatin structures are formed through a two-mode process: initiation (which is context specific) and propagation (which can lead to spreading onto sequences that would not normally be targeted). In our data, H3K9me2 staining is commonly restricted to one side of the synapsed portion of the asymmetric bivalent. This suggests that the propagation of this chromatin mark may require DNA continuity, which would constrain models of how this spreading could occur.

Meiotic crossover redistribution triggered by asymmetric bivalent:

Comparison of crossing over along chromosome IV sequences in control and mnT12/+ males provides insight into the effect of bivalent asymmetry on meiotic recombination. C. elegans males do not carry out germline apoptosis and thus lack a mechanism to eliminate gametes prior to completion of meiosis (Gumienny et al. 1999; Gartner et al. 2000; Jaramillo-Lambert and Engebrecht 2010). In addition, there is no evidence for increased meiotic nondisjunction in mnT12/+ males (Sigurdson et al. 1986; Hillers and Villeneuve 2003). Thus, our observed crossover data likely reflect the actual distribution of crossovers in these animals.

The overall number of chromosome IV crossovers is not significantly reduced in mnT12/+ males (Table 2). However, the distribution of meiotic crossovers along chromosome IV sequences in mnT12/+ males is dramatically different from that seen in control males (Table 3; Figure 3). In control males, 60% of crossovers on chromosome IV occurred in the leftmost 20% of the chromosome, whereas the same region in mnT12/+ males received only 15% of the crossovers. Conversely, in mnT12/+ males, >50% of crossovers now occurred in the rightmost 25% of IV, an interval where only 15% of IV crossovers occurred in control males.

This crossover redistribution is not simply a consequence of the X;IV fusion event that generated mnT12, as the same effect on crossover distribution is not seen in hermaphrodites homozygous for mnT12. In mnT12 hermaphrodites, there is only a slight shift of crossovers away from the left portion of chromosome IV sequences relative to wild type: the crossover frequency for the left 30% of IV in mnT12/mnT12 hermaphrodites was 80% of the control value (data in Hillers and Villeneuve 2003). In mnT12/+ males, there is a much stronger effect: the crossover frequency for roughly the same region in mnT12/+ males was 25% of control levels. This suggests that the observed redistribution of crossovers results in large part from the asymmetric bivalent formed between mnT12 and the unfused chromosome IV. A similar result has been seen in the grasshopper Podisma podestris, in which some populations have a neo-XY male chromosome complement. In these cases, chiasma distribution was also shifted to the fusion-distal portion of the bivalent when compared to populations where the X was not fused to an autosome (John and Hewitt 1970). Taken together, these results suggest that crossover redistribution in response to bivalent asymmetry may be a conserved feature of meiosis.

Meiotic crossing over in C. elegans, as in other eukaryotes, is initiated by meiosis-specific double-strand breaks (DSBs) formed by the enzyme SPO-11. In C. elegans, the overall number of DSBs formed per chromosome is low; in one study, 38% of bivalents appeared to receive a single DSB during meiosis (Mets and Meyer 2009). This indicates that, in C. elegans, crossover distribution may be determined to a surprisingly large extent by DSB position. This suggests that the redistribution of crossovers that we see in mnT12/+ males is accompanied by an altered distribution of DSBs. We can envision two possibilities: It is possible that DSBs themselves are discouraged from occurring near the X;IV fusion point in mnT12/+ males; this could result from the spreading of heterochromatin from X onto conjoined IV sequences in these animals. However, this scenario would imply a discouraging effect of heterochromatin on DSB formation; in light of the recent observation that the single heterochromatic X chromosome in wild-type males receives DSBs, this explanation seems less likely (Jaramillo-Lambert and Engebrecht 2010). Instead, we favor the idea that the altered distribution of crossovers seen in mnT12/+ males requires an overall increase of DSBs along chromosome IV sequences in these animals. We suggest that, in mnT12/+ males, additional DSBs are formed on chromosome IV sequences, with the result that at least one DSB will occur within a region of the chromosome competent for crossing over, i.e., a region engaged in homologous synapsis. A similar explanation likely also accounts for the results of studies examining crossover formation (Zetka and Rose 1992; McKim et al. 1993) and RAD-51 focus levels (Alpi et al. 2003) in C. elegans strains heterozygous for chromosome rearrangements.

Why would additional DSBs form in mnT12/+ males? As previously suggested (Nabeshima et al. 2004; Carlton et al. 2006; Hayashi et al. 2010), our data imply feedback between crossover formation and DSB formation, with the result that DSB formation continues until each bivalent has received the obligate crossover. This model accommodates our results: If the initial DSB on chromosome IV in mnT12/+ males does not result in crossover formation (due to proximity to the X;IV fusion point), DSB formation will continue until a productive (i.e., fusion point-distal) DSB is formed.

What, then, is responsible for the observed crossover redistribution in mnT12/+ males? We favor the idea that the altered crossover distribution is a consequence of synaptic adjustment driving homologous chromosome regions out of register. The synaptic adjustment that we have observed results in the right portion of IV remaining homologously aligned and synapsed, while the left portion becomes misaligned and engages in heterologous synapsis, an arrangement favoring crossovers toward the right and discouraging them toward the left. Furthermore, our time-course analysis indicates that synapsis at IV-L is homologous early in pachytene and becomes heterologous as pachytene progresses. Thus, an important implication of this scenario is that commitment to crossing over would occur relatively late in pachytene, after synaptic adjustment has already taken place.

A neo-pseudoautosomal region:

Sigurdson et al. (1986) first pointed out that mnT12 and IV act as a neo-X and neo-Y, respectively: the homogametic sex (hermaphrodites) is homozygous mnT12, while the heterogametic sex (males) carries mnT12 (the neo-X) and unfused chromosome IV (the neo-Y). Here, we report that crossover distribution along IV is altered in mnT12/+ males, with the majority of crossovers occurring in the right portion of IV (distal to the X;IV fusion point). As noted above, a similar result has been seen in grasshoppers with a neo-XY chromosome complement (John and Hewitt 1970), suggesting that this crossover redistribution may reflect a conserved aspect of meiosis. Here, we consider the effects of such crossover redistribution on sex chromosome evolution.

In a male/female mating system, the suppression of crossovers in fusion-proximal sequences would allow sequence divergence between the neo-X and neo-Y over time. This, in turn, would further discourage crossing over in the fusion-proximal region, resulting in an even stronger shift of crossovers to the distal end of the asymmetric bivalent. However, the meiotic imperative for crossing over (to direct proper disjunction at meiosis I) would lead to continued exchange near the distal end of the asymmetric bivalent, which would prevent divergence of those sequences. Collectively, these features would have the effect of generating a defined region of homology at the distal end of the asymmetric bivalent capable of crossing over, with the remainder of the chromosomes diverged and recombinationally incompatible. This would result in the production of a neo-pseudoautosomal region between the neo-X and the neo-Y (Figure 6). Such a mechanism would also be predicted to result in a gradient of sequence divergence, with regions far from the neo-pseudoautosomal region having the highest levels of divergence. Intriguingly, this is precisely what is seen in three well-studied sex chromosome systems (human X/Y: Skaletsky et al. 2003; chicken Z/W: Handley et al. 2004; Silene latifolia X/Y: Bergero et al. 2007).

Figure 6.—

Figure 6.—

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Generation of a neo-pseudoautosomal region. In neo-X/neo-Y males, bivalent asymmetry leads to a redistribution of crossovers toward the fusion-distal portion of the asymmetric bivalent, with a corresponding reduction of crossovers near the fusion point (top). Over time, this will allow sequence divergence between the neo-Y and neo-X near the fusion point, which will further reduce the probability of crossing over in fusion-proximal sequences (middle). Eventually, sequence divergence between the fusion-proximal portions of the neo-X and neo-Y will present a barrier to exchange. However, the meiotic imperative for crossover formation will prevent sequence divergence at the distal end of the chromosomes. As a result, the fusion-distal end of the neo-X and neo-Y will function as a neo-pseudoautosomal region (PAR; bottom).

Our data suggest that bivalent asymmetry in combination with synaptic adjustment can directly result in restriction of meiotic crossovers to a limited portion of the bivalent, which could then promote diversification of neo-sex chromosomes without need for subsequent inversions to inhibit crossing over. Thus, we suggest that chromosome fusion events that generate asymmetric bivalents can lead to fundamental alterations in the recombinational landscape of chromosomes and can potentially contribute to the evolution of sex chromosomes.

Acknowledgments

We thank the Caenorhabditis Genetics Center for strains; M. Zetka for HIM-3 antibodies; Anna Naccarati for technical assistance; and members of the Villeneuve lab for technical advice and helpful discussions. This work was supported in part by National Institutes of Health grants R15HD059093 to K.J.H. and R01GM67268 to A.M.V.; by research grant #5-FY07-666 from the March of Dimes Foundation to K.N.; by a Canadian Institute of Health Research postdoctoral fellowship to M.S.; and by the Cal Poly College-Based Fee program.

References

  1. Albertson, D. G., A. M. Rose and A. M. Villeneuve, 1997. Chromosome organization, mitosis, and meiosis, pp. 47–78 in C. elegans II, edited by D. L. Riddle, T. Blumenthal, B. J. Meyer and J. R. Priess. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [PubMed]
  2. Alpi, A., P. Pasierbek, A. Gartner and J. Loidl, 2003. Genetic and cytological characterization of the recombination protein RAD-51 in Caenorhabditis elegans. Chromosoma 112 6–16. [DOI] [PubMed] [Google Scholar]
  3. Baarends, W. M., E. Wassenaar, R. van der Laan, J. Hoogerbrugge, E. Sleddens-Linkels et al., 2005. Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis. Mol. Cell. Biol. 25 1041–1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Barnes, T. M., Y. Kohara, A. Coulson and S. Hekimi, 1995. Meiotic recombination, noncoding DNA and genomic organization in Caenorhabditis elegans. Genetics 141 159–179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bean, C. J., C. E. Schaner and W. G. Kelly, 2004. Meiotic pairing and imprinted X chromatin assembly in Caenorhabditis elegans. Nat. Genet. 36 100–105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bergero, R., and D. Charlesworth, 2009. The evolution of restricted recombination in sex chromosomes. Trends Ecol. Evol. 24 94–102. [DOI] [PubMed] [Google Scholar]
  7. Bergero, R., A. Forrest, E. Kamau and D. Charlesworth, 2007. Evolutionary strata on the X chromosomes of the dioecious plant Silene latifolia: evidence from new sex-linked genes. Genetics 175 1945–1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brenner, S., 1974. The genetics of Caenorhabditis elegans. Genetics 77 71–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carlton, P. M., A. P. Farruggio and A. F. Dernburg, 2006. A link between meiotic prophase progression and crossover control. PLoS Genet. 2 e12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Couteau, F., and M. Zetka, 2005. HTP-1 coordinates synaptonemal complex assembly with homolog alignment during meiosis in C. elegans. Genes Dev. 19 2744–2756. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ding, D. Q., T. Haraguchi and Y. Hiraoka, 2010. From meiosis to postmeiotic events: alignment and recognition of homologous chromosomes in meiosis. FEBS J. 277 565–570. [DOI] [PubMed] [Google Scholar]
  12. Edgar, L. G., 1995. Blastomere culture and analysis. Methods Cell Biol. 48 303–321. [DOI] [PubMed] [Google Scholar]
  13. Ercan, S., L. L. Dick and J. D. Lieb, 2009. The C. elegans dosage compensation complex propagates dynamically and independently of X chromosome sequence. Curr. Biol. 19 1777–1787. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gartner, A., S. Milstein, S. Ahmed, J. Hodgkin and M. O. Hengartner, 2000. A conserved checkpoint pathway mediates DNA damage–induced apoptosis and cell cycle arrest in C. elegans. Mol. Cell 5 435–443. [DOI] [PubMed] [Google Scholar]
  15. Gonczy, P., S. Pichler, M. Kirkham and A. A. Hyman, 1999. Cytoplasmic dynein is required for distinct aspects of MTOC positioning, including centrosome separation, in the one cell stage Caenorhabditis elegans embryo. J. Cell Biol. 147 135–150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gumienny, T. L., E. Lambie, E. Hartwieg, H. R. Horvitz and M. O. Hengartner, 1999. Genetic control of programmed cell death in the Caenorhabditis elegans hermaphrodite germline. Development 126 1011–1022. [DOI] [PubMed] [Google Scholar]
  17. Handley, L. J., H. Ceplitis and H. Ellegren, 2004. Evolutionary strata on the chicken Z chromosome: implications for sex chromosome evolution. Genetics 167 367–376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hayashi, M., S. Mlynarczyk-Evans and A. M. Villeneuve, 2010. The synaptonemal complex shapes the crossover landscape through cooperative assembly, crossover promotion and crossover inhibition during Caenorhabditis elegans meiosis. Genetics 186 45–58. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Herman, R. K., and C. K. Kari, 1989. Recombination between small X chromosome duplications and the X chromosome in Caenorhabditis elegans. Genetics 121 723–737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hillers, K. J., and A. M. Villeneuve, 2003. Chromosome-wide control of meiotic crossing over in C. elegans. Curr. Biol. 13 1641–1647. [DOI] [PubMed] [Google Scholar]
  21. Hillers, K. J., and A. M. Villeneuve, 2009. Analysis of meiotic recombination in Caenorhabditis elegans. Methods Mol. Biol. 557 77–97. [DOI] [PubMed] [Google Scholar]
  22. Jaramillo-Lambert, A., and J. Engebrecht, 2010. A single unpaired and transcriptionally silenced X chromosome locally precludes checkpoint signaling in the Caenorhabditis elegans germ line. Genetics 184 613–628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. John, B., and G. M. Hewitt, 1970. Inter-population sex chromosome polymorphism in the grasshopper Podisma pedestris. I. Fundamental facts. Chromosoma 31 291–308. [DOI] [PubMed] [Google Scholar]
  24. Keeney, S., 2001. Mechanism and control of meiotic recombination initiation. Curr. Top. Dev. Biol. 52 1–53. [DOI] [PubMed] [Google Scholar]
  25. Kelly, W. G., C. E. Schaner, A. F. Dernburg, M. H. Lee, S. K. Kim et al., 2002. X-chromosome silencing in the germline of C. elegans. Development 129 479–492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kleckner, N., 1996. Meiosis: How could it work? Proc. Natl. Acad. Sci. USA 93 8167–8174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. MacQueen, A. J., M. P. Colaiacovo, K. McDonald and A. M. Villeneuve, 2002. Synapsis-dependent and -independent mechanisms stabilize homolog pairing during meiotic prophase in C. elegans. Genes Dev. 16 2428–2442. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. MacQueen, A. J., C. M. Phillips, N. Bhalla, P. Weiser, A. M. Villeneuve et al., 2005. Chromosome sites play dual roles to establish homologous synapsis during meiosis in C. elegans. Cell 123 1037–1050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mahadevaiah, S. K., J. M. Turner, F. Baudat, E. P. Rogakou, P. de Boer et al., 2001. Recombinational DNA double-strand breaks in mice precede synapsis. Nat. Genet. 27 271–276. [DOI] [PubMed] [Google Scholar]
  30. Martinez-Perez, E., and A. M. Villeneuve, 2005. HTP-1-dependent constraints coordinate homolog pairing and synapsis and promote chiasma formation during C. elegans meiosis. Genes Dev. 19 2727–2743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Martinez-Perez, E., M. Schvarzstein, C. Barroso, J. Lightfoot, A. F. Dernburg et al., 2008. Crossovers trigger a remodeling of meiotic chromosome axis composition that is linked to two-step loss of sister chromatid cohesion. Genes Dev. 22 2886–2901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McKim, K. S., A. M. Howell and A. M. Rose, 1988. The effects of translocations on recombination frequency in Caenorhabditis elegans. Genetics 120 987–1001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. McKim, K. S., K. Peters and A. M. Rose, 1993. Two types of sites required for meiotic chromosome pairing in Caenorhabditis elegans. Genetics 134 749–768. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mets, D. G., and B. J. Meyer, 2009. Condensins regulate meiotic DNA break distribution, thus crossover frequency, by controlling chromosome structure. Cell 139 73–86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mlynarczyk-Evans, S., and A. M. Villeneuve, 2010. Homologous chromosome pairing and synapsis during oogenesis, pp. 117–140 in Oogenesis: The Universal Process, edited by M.-H. Verlhac and A. M. Villeneuve. John Wiley & Sons, Chichester, UK.
  36. Moses, M. J., and P. A. Poorman, 1981. Synaptosomal complex analysis of mouse chromosomal rearrangements. II. Synaptic adjustment in a tandem duplication. Chromosoma 81 519–535. [DOI] [PubMed] [Google Scholar]
  37. Nabeshima, K., A. M. Villeneuve and K. J. Hillers, 2004. Chromosome-wide regulation of meiotic crossover formation in Caenorhabditis elegans requires properly assembled chromosome axes. Genetics 168 1275–1292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nabeshima, K., A. M. Villeneuve and M. P. Colaiacovo, 2005. Crossing over is coupled to late meiotic prophase bivalent differentiation through asymmetric disassembly of the SC. J. Cell Biol. 168 683–689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Oegema, K., A. Desai, S. Rybina, M. Kirkham and A. A. Hyman, 2001. Functional analysis of kinetochore assembly in Caenorhabditis elegans. J. Cell Biol. 153 1209–1226. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Page, S. L., and R. S. Hawley, 2001. (3)G encodes a Drosophila synaptonemal complex protein. Genes Dev. 15 3130–3143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Page, S. L., and R. S. Hawley, 2003. Chromosome choreography: the meiotic ballet. Science 301 785–789. [DOI] [PubMed] [Google Scholar]
  42. Penkner, A., L. Tang, M. Novatchkova, M. Ladurner, A. Fridkin et al., 2007. The nuclear envelope protein Matefin/SUN-1 is required for homologous pairing in C. elegans meiosis. Dev. Cell 12 873–885. [DOI] [PubMed] [Google Scholar]
  43. Penkner, A. M., A. Fridkin, J. Gloggnitzer, A. Baudrimont, T. Machacek et al., 2009. Meiotic chromosome homology search involves modifications of the nuclear envelope protein Matefin/SUN-1. Cell 139 920–933. [DOI] [PubMed] [Google Scholar]
  44. Petronczki, M., M. F. Siomos and K. Nasmyth, 2003. Un menage a quatre: the molecular biology of chromosome segregation in meiosis. Cell 112 423–440. [DOI] [PubMed] [Google Scholar]
  45. Phillips, C. M., and A. F. Dernburg, 2006. A family of zinc-finger proteins is required for chromosome-specific pairing and synapsis during meiosis in C. elegans. Dev. Cell 11 817–829. [DOI] [PubMed] [Google Scholar]
  46. Phillips, C. M., C. Wong, N. Bhalla, P. M. Carlton, P. Weiser et al., 2005. HIM-8 binds to the X chromosome pairing center and mediates chromosome-specific meiotic synapsis. Cell 123 1051–1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Phillips, C. M., X. Meng, L. Zhang, J. H. Chretien, F. D. Urnov et al., 2009. Identification of chromosome sequence motifs that mediate meiotic pairing and synapsis in C. elegans. Nat. Cell Biol. 11 934–942. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Reinke, V., H. E. Smith, J. Nance, J. Wang, C. Van Doren et al., 2000. A global profile of germline gene expression in C. elegans. Mol. Cell 6 605–616. [DOI] [PubMed] [Google Scholar]
  49. Rockman, M. V., and L. Kruglyak, 2009. Recombinational landscape and population genomics of Caenorhabditis elegans. PLoS Genet. 5 e1000419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Rosenbluth, R. E., and D. L. Baillie, 1981. The genetic analysis of a reciprocal translocation, eT1(III; V), in Caenorhabditis elegans. Genetics 99 415–428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Sato, A., B. Isaac, C. M. Phillips, R. Rillo, P. M. Carlton et al., 2009. Cytoskeletal forces span the nuclear envelope to coordinate meiotic chromosome pairing and synapsis. Cell 139 907–919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Schoenmakers, S., E. Wassenaar, J. W. Hoogerbrugge, J. S. Laven, J. A. Grootegoed et al., 2009. Female meiotic sex chromosome inactivation in chicken. PLoS Genet. 5 e1000466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Shakes, D. C., J. C. Wu, P. L. Sadler, K. Laprade, L. L. Moore et al., 2009. Spermatogenesis-specific features of the meiotic program in Caenorhabditis elegans. PLoS Genet. 5 e1000611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sigurdson, D. C., R. K. Herman, C. A. Horton, C. K. Kari and S. E. Pratt, 1986. An X-autosome fusion chromosome of Caenorhabditis elegans. Mol. Gen. Genet. 202 212–218. [DOI] [PubMed] [Google Scholar]
  55. Skaletsky, H., T. Kuroda-Kawaguchi, P. J. Minx, H. S. Cordum, L. Hillier et al., 2003. The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes. Nature 423 825–837. [DOI] [PubMed] [Google Scholar]
  56. Sym, M., and G. S. Roeder, 1994. Crossover interference is abolished in the absence of a synaptonemal complex protein. Cell 79 283–292. [DOI] [PubMed] [Google Scholar]
  57. Turner, J. M., S. K. Mahadevaiah, P. J. Ellis, M. J. Mitchell and P. S. Burgoyne, 2006. Pachytene asynapsis drives meiotic sex chromosome inactivation and leads to substantial postmeiotic repression in spermatids. Dev. Cell 10 521–529. [DOI] [PubMed] [Google Scholar]
  58. Villeneuve, A. M., 1994. A cis-acting locus that promotes crossing over between X chromosomes in Caenorhabditis elegans. Genetics 136 887–902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wilson, M. A., and K. D. Makova, 2009. Genomic analyses of sex chromosome evolution. Annu. Rev. Genomics Hum. Genet. 10 333–354. [DOI] [PubMed] [Google Scholar]
  60. Zetka, M. C., and A. M. Rose, 1992. The meiotic behavior of an inversion in Caenorhabditis elegans. Genetics 131 321–332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zetka, M. C., I. Kawasaki, S. Strome and F. Muller, 1999. Synapsis and chiasma formation in Caenorhabditis elegans require HIM-3, a meiotic chromosome core component that functions in chromosome segregation. Genes Dev. 13 2258–2270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zickler, D., and N. Kleckner, 1999. Meiotic chromosomes: integrating structure and function. Annu. Rev. Genet. 33 603–754. [DOI] [PubMed] [Google Scholar]

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