The Mouse Spo11 Gene Is Required for Meiotic Chromosome Synapsis

The Mouse Spo11 Gene Is Required for Meiotic Chromosome Synapsis

Molecular Cell, Vol. 6, 975–987, November, 2000, Copyright 2000 by Cell Press The Mouse Spo11 Gene Is Required for Meiotic Chromosome Synapsis Peter...

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Molecular Cell, Vol. 6, 975–987, November, 2000, Copyright 2000 by Cell Press

The Mouse Spo11 Gene Is Required for Meiotic Chromosome Synapsis Peter J. Romanienko and R. Daniel Camerini-Otero* Genetics and Biochemistry Branch National Institute of Diabetes and Digestive and Kidney Diseases National Institutes of Health Bethesda, Maryland 20892

Summary The Spo11 protein initiates meiotic recombination by generating DNA double-strand breaks (DSBs) and is required for meiotic synapsis in S. cerevisiae. Surprisingly, Spo11 homologs are dispensable for synapsis in C. elegans and Drosophila yet required for meiotic recombination. Disruption of mouse Spo11 results in infertility. Spermatocytes arrest prior to pachytene with little or no synapsis and undergo apoptosis. We did not detect Rad51/Dmc1 foci in meiotic chromosome spreads, indicating DSBs are not formed. Cisplatin-induced DSBs restored Rad51/Dmc1 foci and promoted synapsis. Spo11 localizes to discrete foci during leptotene and to homologously synapsed chromosomes. Other mouse mutants that arrest during meiotic prophase (Atm ⫺/⫺, Dmc1 ⫺/⫺, mei1, and Morc⫺/⫺) showed altered Spo11 protein localization and expression. We speculate that there is an additional role for Spo11, after it generates DSBs, in synapsis. Introduction Sexual reproduction and meiotic recombination are common to most eukaryotes (Welch and Meselson, 2000). Meiosis is a specialized cell division that reduces the normal chromosome complement by half in order to generate haploid gametes. The proper segregation of chromosomes into gametes generally requires meiotic recombination. In most organisms, genetic exchange occurs, and homologous chromosomes undergo synapsis and form a synaptonemal complex (SC) (Heyting, 1996). The SC is a proteinaceous structure composed of lateral elements (LE) and the central element (CE). The LEs are parallel structures that originate as the axial elements (AE), one component of which is the protein Scp3 (Dobson et al., 1994). The AEs are seen at leptotene during meiotic prophase I and eventually form along the entire length of each homolog. Synapsis can be visualized when two AEs of homologous chromosomes come together during zygotene (Zickler and Kleckner, 1998). The CE is formed when transverse filaments (TFs), composed of the protein Scp1, bridge the axial elements during zygotene and pachytene (Schmekel and Daneholt, 1998). Whereas the initiation of meiotic recombination appears to be highly conserved, some eukaryotes differ in the molecular requirements for synapsis (Walker and * To whom correspondence should be addressed (e-mail: [email protected] ncifcrf.gov).

Hawley, 2000). For instance, meiotic recombination is initiated by DNA DSBs generated by the action of the Spo11 protein (Spo11p) in S. cerevisiae (Keeney et al., 1997), and in the absence of Spo11p, DSBs are not formed and homologous chromosomes do not synapse at wild-type levels (Giroux et al., 1989). Similarly, the disruption of SPO11 homologs in S. pombe (Cervantes et al., 2000), Drosophila (McKim et al., 1998), C. elegans (Dernburg et al., 1998), and Coprinus cinereus (Celerin et al., 2000) results in defective gametes, and a reduction in meiotic recombination has been reported in S. cerevisiae, S. pombe, Drosophila, and C. elegans. Chromosomes still synapse at wild-type levels in spo11⫺/⫺ mutants of the worm and fly but not in Coprinus cinereus. In addition, meiosis is partially rescued by radiationinduced DSBs in budding yeast (Thorne and Byers, 1993; Gasior, 1999), worm (Dernburg et al., 1998), fly (R. Patel and K. McKim, personal communication), and Coprinus cinereus (Celerin et al., 2000). These rescue experiments provide evidence for meiotic DSB formation in eukaryotes other than S. cerevisiae and S. pombe (Cervantes et al., 2000) and that the requirement for Spo11 can be partially bypassed. Thus, it appears that in worm and fly, synapsis can be mediated by pairing centers that function independently of the need for meiotic recombination. If dependence on pairing centers is seen with an increase in genome complexity, one might easily imagine that mammals might also utilize pairing centers. Spo11-mediated DSBs occur at leptotene in S. cerevisiae and disappear by zygotene (Roeder, 1997). This coincides with localization of Rad51 and Dmc1, two eukaryotic RecA homologs, to discrete foci in mouse (Tarsounas et al., 1999) and in a Spo11-dependent manner in yeast (Bishop, 1994), the former presumably also at sites of DSBs. The presence of Rad51 and Dmc1 is consistent with their biochemical function in DNA strand exchange at the site of DNA DSBs. Spo11p is a member of a novel family of type II-like topoisomerases, and consistent with that, site-directed mutagenesis of a conserved tyrosine (Y135F) abolishes meiotic recombination (Bergerat et al., 1997). Homologs of Spo11p have been isolated from mouse, Spo11, (Keeney et al., 1999; Romanienko and Camerini-Otero, 1999; Shannon et al., 1999; Metzler-Guillemain and de Massy, 2000) and man, SPO11, (Romanienko and Camerini-Otero, 1999; Shannon et al., 1999), and their expression patterns are consistent with having a meiotic function. Using RT-PCR, many alternatively spliced variants can be detected in testes, and Spo11 transcripts are also detected in somatic tissues. Analysis of Spo11 expression in staged mouse spermatocytes showed the highest level of transcript during pachytene (Shannon et al., 1999), which is unexpected with Spo11 catalyzing DNA DSBs during leptotene. This would be more consistent with an additional role for Spo11 when homologous chromosomes are fully synapsed. In fact, SPO11 transcript levels are also at their highest levels during pachytene in yeast (Atcheson et al., 1987; Chu et al., 1998), but no late function for Spo11p has been proposed.

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Figure 1. Generation Mice

of

Spo11-Deficient

(A) Gene disruption of the Spo11 locus. The targeting construct and genomic locus of Spo11 are shown. The EcoRI site in intron 1 was disrupted, and another EcoRI site was created at the junction of the neo cassette and exon 1 of Spo11 to facilitate identification of targeted clones. The probe was derived from the Spo11 cDNA encompassing exons 10–13. An EcoRI digest would give a 13 kb band from wild-type genomic DNA and a 16 kb band from a targeted allele. (B) Southern blot analysis of representative genotypes of Spo11 knockout mice. Homozygous knockout, ⫺/⫺; heterozygous, ⫹/⫺; wild-type, ⫹/⫹. The digest was done with EcoRI and probed with Spo11 cDNA, exons 10–13. The arrows indicate positions of bands and their sizes are indicated. (C) PCR genotyping of Spo11 knockout mice by PCR. Mice used in experiments were genotyped by PCR. The same mice were used for Southern blot analysis in (B), indicating the method is consistent with results from Southern blotting. The primer locations are indicated in (A). Targeting at the Spo11 locus removes the binding site for one Spo11-specific primer. (D) Northern analysis of Spo11 knockout mice. Poly(A)⫹ RNA isolated from testes of mice with the indicated genotypes was probed with the mouse coding sequence cDNA, and the same blot was later probed with the mouse ␤-actin cDNA. The Spo11 transcript (1.8 kb) was absent in ⫺/⫺ testes and reduced in the heterozygote. The ␤-actin signal is not relevant to the phenotype and serves as a loading standard. Postmeiotic ␣-actin was absent in ⫺/⫺ testes.

Here, we disrupted the mouse Spo11 gene and determined it is essential for gametogenesis and meiotic chromosome synapsis. In addition, we found that Spo11 localizes to discrete foci early in meiosis and later to regions of homologous chromosome synapsis. Furthermore, Spo11 expression and localization of the protein is altered in mutant mice that arrest during meiotic prophase I. Finally, we were able to partially rescue the defect in synapsis in Spo11⫺/⫺ spermatocytes using the DSB-causing reagent cisplatin. Results Targeted Inactivation of Mouse Spo11 The mouse Spo11 gene was inactivated by gene targeting in embryonic stem (ES) cells. A targeting construct was made (Figure 1A) that removed part of the coding sequence of exon 1 from 3 amino acids after the second in-frame ATG up to nucleotide 35 of the first intron (Keeney et al., 1999; Romanienko and CameriniOtero, 1999). The removal of most of exon 1 and part of intron 1 was designed to effectively create a protein null without greatly disturbing the genomic locus. Targeted clones were analyzed by Southern blotting, and a correctly targeted cell line was injected into blastocysts. Southern blot analysis of representative wild-type, heterozygotic, and double knockout mice (Figure 1B) is shown. The ability to generate homozygous knockout mice indicates Spo11 is not essential for mouse development. The mutated Spo11 allele was transmitted to

offspring in a Mendelian fashion, and homozygotic knockout mice did not exhibit any gross external morphological defects. Mice used for experiments were genotyped by PCR on genomic DNA (Figure 1C). Northern blot analysis of mRNA from adult testes from Spo11 ⫹/⫹, ⫹/⫺, and ⫺/⫺ mice (Figure 1D) showed that the transcript was reduced in heterozygotes and absent from ⫺/⫺ testes. The same blot was probed with mouse ␤-actin as a control for the loading of mRNA. Mouse ␤-actin cross-hybridizes to postmeiotically expressed ␣-actin (Hecht et al., 1984). The absence of ␣-actin in Spo11⫺/⫺ testes is evidence that there is an arrest prior to the appearance of postmeiotic cells. These results show that the Spo11 gene is inactivated in ⫺/⫺ mice. Spo11⫺/⫺ Male Mice Are Infertile The first indication of a defect attributable to Spo11 deficiency is the significant decrease in testicular size in mutant males (Figure 2A). The reduced size of the testis is similar to results seen in other mouse knockouts of early meiotic genes (Yoshida et al., 1998; Yuan et al., 2000). Histological analysis of seminiferous tubule cross sections from wild-type (Figure 2B) and mutant mice show many of the tubules in the mutants are barren, indicating massive cell death or nonproliferation, while other tubules show clusters of cells morphologically similar to zygotene spermatocytes (Figure 2C). There were no cells with pachytene morphology in Spo11⫺/⫺ tubules. Spermatocytes in Spo11⫺/⫺ mice underwent apoptosis (Figure 2E), while few apoptotic cells were

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Figure 2. Histological Examination of Adult Testes from Spo11⫺/⫺ Mice (A) Reduced testicular size in Spo11⫺/⫺ mice. Wild-type, ⫹/⫹; heterozygote, ⫹/⫺; and homozygote knockout, ⫺/⫺ testes were compared from 6-week-old male sibs. (B) Hematoxylin- and eosin-stained cross section of a seminiferous tubule from a wild-type adult mouse. Magnification is 400⫻. (C) Hematoxylin- and eosin-stained cross sections of seminiferous tubules from a Spo11⫺/⫺ adult mouse. Magnification is 400⫻. (D) Fluorescent TUNEL assay in wild-type mouse testis section; labeling was detected in some tubules near the basal lamina (not shown). Magnification is 200⫻. (E) Fluorescent TUNEL assay in Spo11⫺/⫺ mouse testis section; labeled cells (green fluorescence within tubules) were detected in about 20% of tubules. Magnification is 200⫻; the arrow indicates positive tubule.

detected in wild-type mice (Figure 2D). Approximately 20% of the tubules in the mutant had clusters of apoptotic cells, while wild-type mice only showed an occasional apoptotic cell. Spo11⫺/⫺ Female Mice Are Infertile Histological cross sections of wild-type and mutant adult ovaries showed a reduction in the number of follicles in the mutant (Figure 3A, wild type; Figure 3B, ⫺/⫺), indicating there is a severe phenotype in female mice resulting in few or no mature oocytes. Defects can be seen as early as 15 dpc in the fetal ovaries of ⫺/⫺ mice (Figure 3D) that are entering the early stages of meiosis I. In comparison with wild-type mice (Figure 3C), the cells in the mutant fetal ovary look homogeneous. Cells with partially condensed chromatin, indicating zygotene cells in wild-type mice, were reduced in the absence of Spo11 (Figure 3D). We were unsuccessful in obtaining

pregnant Spo11⫺/⫺ mice after several months of breeding with wild-type male mice. Spermatocytes from Spo11⫺/⫺ Mice Arrest Prior to Pachytene Spermatocytes from Spo11⫺/⫺ mice proceed normally through leptotene (Figure 4A), and 40 centromeres are visualized by staining with CREST antisera, which labels centromeres (He and Brinkley, 1996). The presence of 40 centromeres at this early stage indicates there is no disruption of sister chromatid cohesion at the centromeres in the absence of Spo11. Most spermatocytes arrest later at a zygotene-like stage (Figure 4B) where there is little or no synapsis between chromosomes, whether homologous or nonhomologous. While the meiotic arrest is absolute, the failure to synapse is not. Approximately 5%–10% of the spread nuclei exhibit partial, and in some instances, considerable synapsis (Fig-

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Figure 3. Histological Examination of Adult and Fetal Ovaries from Spo11⫺/⫺ Mice (A) Hematoxylin- and eosin-stained adult wild-type ovary. Arrow indicates maturing follicle. Eight to twelve follicles were generally seen per section. Magnification is 200⫻. (B) Hematoxylin- and eosin-stained adult Spo11⫺/⫺ ovary. Arrow indicates follicle, but note absence of any other follicles. No more than one or two follicles were seen per section. Magnification is 200⫻. (C) Hematoxylin- and eosin-stained wild-type fetal ovary, 15 dpc. The two female fetal mice were from the same dam. Arrows denote cells with condensed chromatin. Magnification is 1000⫻. (D) Hematoxylin- and eosin-stained Spo11⫺/⫺ fetal ovary, 15 dpc. Note fewer cells with condensed chromatin morphology as compared to (C). The number of cells with this morphology was half of those seen in wild type. Magnification is 1000⫻.

ure 4G). The synapsis, indicated by Scp3 staining of lateral elements, occurs mostly between nonhomologs since frequent partner switches are observed. Scp3 staining of lateral elements colocalized with Scp1 staining (unpublished observations). Rad51 and Dmc1 are RecA-homologs, the latter meiosis-specific, that are required for strand invasion (Shinohara et al., 1997). Rad51 and Dmc1 are believed to load onto single-stranded DNA generated at the sites of meiotic DSBs. Neither Rad51 nor Dmc1 foci were seen in Spo11⫺/⫺ spermatocytes (Figures 4D and 4F), but they were readily detected in wild-type spermatocytes (Figure 4C and 4E). The ␣Rad51 antibody routinely detected one or two large signals in Spo11⫺/⫺ spermatocytes. These signals may be cellular reservoirs of Rad51 or cross-reactivity to another protein. We do not believe the latter is true, since we do not see this staining in wild-type nuclei. The absence of Rad51 and Dmc1 foci confirms that meiotic DSBs are not formed in the absence of Spo11.

Localization of Mouse Spo11 in Meiotic Chromosome Spreads from Spermatocytes Some meiotic proteins can be visualized with respect to meiotic chromosomes, and this approach often yields insights into what role a specific protein may play during meiosis (Plug et al., 1998; Moens et al., 1999). Spo11 has not been visualized in meiotic nuclei of any organism previously. We performed indirect immunofluorescence of meiotic chromosome spreads using an affinity-purified antibody raised against a peptide (MmPep-1) derived from the Toprim domain (Aravind et al., 1998) of the mouse Spo11 protein. First, we performed several controls to make sure the antisera and purified antibody were Spo11 specific. Preimmune sera did not recognize SCs in meiotic spreads, but immune serum did. The signal from crude antiserum and purified antibody was abolished by incubation in the presence of 10-fold molar excess of purified MmPep-1 but not in the presence of 100-fold molar excess of an unrelated peptide. Rabbit ␣-mouse Spo11 was able to detect the two major forms

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Figure 4. Indirect Immunofluorescence of Spo11⫺/⫺ Spermatocytes (A) Leptotene stage Spo11⫺/⫺ spermatocyte. Merged image of ␣Scp3 (red) and the centromere antisera CREST (green). All images initially viewed at 1000⫻ magnification. Scale bar, 10 ␮m. Overlap of red and green signals results in yellow signal. (B) Zygotene-like stage of arrest in Spo11⫺/⫺ spermatocytes. Merged image of ␣Scp3 (red) and the centromere antisera CREST (green). Three nuclei are shown. (C) Zygotene wild-type spermatocyte; merged image of ␣Scp3 (red) and the ␣Rad51 (green). (D) Zygotene-like Spo11⫺/⫺ spermatocyte; merged image of ␣Scp3 (red) and the ␣Rad51 (green). (E) Zygotene wild-type spematocyte; merged image of ␣Scp3 (red) and the ␣Dmc1 (green). (F) Zygotene-like Spo11⫺/⫺ spermatocyte; merged image of ␣Scp3 (red) and the ␣Dmc1 (green). (G) Zygotene-like Spo11⫺/⫺ spematocyte showing extent of synapsis, indicated by arrows. Stained with ␣Scp3 (red).

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Figure 5. Spo11 Localization in Meiotic Chromosome Spreads (A) Spo11⫺/⫺ spermatocyte; merged image of ␣Scp3 (red) and the ␣Spo11 (green). Note absence of Spo11 signal. All images were initially viewed at 1000⫻ magnification. Scale bar, 10 ␮m. Overlap of red and green signals results in yellow signal. (B) Leptotene spermatocyte; merged image of ␣Scp3 (red) and the ␣Spo11 (green). (C) Zygotene spermatocyte; merged image of ␣Scp3 (red) and the ␣Spo11 (green). Arrows indicate regions of synapsis. (D) Pachytene spermatocyte; merged image of ␣Scp3 (red) and the ␣Spo11 (green). Arrow indicates the pseudoautosomal region (PAR). (E) Magnification of (C); arrow indicates PAR. (F) Pachytene spermatocyte; merged image of ␣Scp1 (red) and the ␣Spo11 (green). Arrow indicates PAR. (G) Magnification of (E); arrow indicates PAR.

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Figure 6. Spo11 Localization and Expression in Mouse Meiotic Mutants (A) RT-PCR analysis of Spo11 expression in testes of mutant mice. The two forms of Spo11 (␣ and ␤) are indicated by arrows in the top panel, and the bottom panel shows control amplification of Aop2 (Pittman et al., 1998). The mutants analyzed are listed across the top. 13.5 dpp (days postpartum), amplification from testis cDNA from a juvenile mouse; H20, water control in amplification reaction. ␣ and ␤ forms of Spo11 are shown with primer location relative to exon 2, active site region, and Toprim domain. The sizes of putative proteins are indicated on the right. aa, amino acids. (B) Localization of Spo11 in Dmc1⫺/⫺ spermatocyte. Merged image of ␣Scp3 (red) and ␣Spo11 (green). Arrowhead indicates Spo11 staining in synapsed region, arrows indicate ends of axial elements of homologous chromosome participating in homologous synapsis, and asterisks indicate synapsis between nonhomologs. Images were initially viewed at 1000⫻ magnification. The scale bar is 10 ␮m. Overlap of red and green signals results in yellow signal. (C) Localization of Spo11 in mei1 spermatocytes. Merged image of ␣Scp3 (red) and ␣Spo11 (green). Arrow indicates Spo11 staining of fully synapsed homologs, and asterisks indicate synapsis between nonhomologs. Two nuclei are shown.

of Spo11 expressed from expression vectors transfected into mouse 3T3 cells but did not react with any other protein from untransfected 3T3 cells (data not shown). The inability to visualize Spo11 foci or localization at partially synapsed regions (Figure 5A) in Spo11⫺/⫺ mice is strong evidence that our antibody to Spo11 is specific. We first detected Spo11 during the leptotene stage of meiotic prophase I (Figure 5B). This is the stage when meiotic DSBs are generated in budding yeast (Roeder, 1997). The majority of Spo11 foci (green) did not colocalize with the antibody marker (␣Scp3, red) we used for assessing the extent of axial element formation. These foci most likely represent sites of DNA DSB formation.

The next stage in meiosis is zygotene, during which homologous chromosomes begin to synapse (Zickler and Kleckner, 1998). The distribution of Spo11 changes, and localization is now to the regions of synapsis between homologs (Figure 5C). Full synapsis of homologs occurs during pachytene, and Spo11 decorates the lengths of all autosomes (stained with ␣Scp3) in a discontinuous pattern (Figure 5D). ␣Spo11 also stains the XY chromosome pair only at the pseudoautsomal region (PAR) (Figure 5D), where the X and Y chromosomes homologously synapse and recombine in the male (Soriano et al., 1987). A magnification of this region (Figure 5E) shows the PAR and the discontinuous localization of Spo11 in more detail. Spo11 colocalized with the

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SC, as determined by Scp1 staining (Figure 5F). Similar results are seen in a magnification of the PAR from Figure 5F (Figure 5G). Spo11 dissociates from the SC during diplotene as homologs begin to separate. Before desynapsis is complete, all Spo11 localization with the SC vanishes and staining becomes diffuse (data not shown). Spo11 Expression and Localization in Spermatocytes from Other Mutant Mice Arrested in Meiotic Prophase I In order to gain further insight into the roles Spo11 may have in meiosis, we looked at Spo11 expression and localization in several mutant backgrounds. Atm⫺/⫺ mice are deficient for the mouse homolog of the gene mutated in ataxia-telangiectasia (AT) patients that have a defect in DNA repair and are infertile (Barlow et al., 1996). Atm⫺/⫺ spermatocytes also show mislocalization of Rad51 and Dmc1 to chromatin and reduced localization of these proteins to the developing SC (Barlow et al., 1998). Dmc1⫺/⫺ mice are mutant for the meiosis-specific mouse RecA homolog, Dmc1 (Pittman et al., 1998), and arrest but show normal localization of Rad51. The presence of Rad51 foci in both Atm⫺/⫺ and Dmc1⫺/⫺ spermatocytes is evidence that meiotic DSBs are made in these mutants. mei1 mice were derived from mutagenized ES cells and are infertile (Munroe et al., 2000). Morc⫺/⫺ mice are the result of a random transgene insertion (Watson et al., 1998), and the mutated gene encodes a protein with limited homology to GHL (GyraseB, Hsp90, Mut L) ATPases (Inoue et al., 1999). In mouse and man there are two predominant, alternatively spliced forms of Spo11 (Romanienko and Camerini-Otero, 1999; Shannon et al., 1999) that code for two proteins of different sizes (unpublished data). RT-PCR analysis of Spo11 expression in a panel of mutant mice revealed a common defect in generation of the predominant spliced form of the transcript (Figure 6A). All mice showed a severe reduction in removal of exon 2 (Figure 6A, compare lanes 1–3 with lane 5). The splicing of another transcript, DNA ligase III, was analyzed in these mutant mice. Splicing of the ␤ form of DNA ligase III occurs in pachytene spermatocytes (Mackey et al., 1997), but we saw no difference in the splicing pattern of this transcript in the mutant mice we analyzed (data not shown). The localization of mouse Spo11 was performed in meiotic chromosome spreads from mouse mutants that arrest during meiotic prophase I. The mouse mutants used, for the most part, do not show complete homologous synapsis. The distribution of Spo11 was different in Dmc1⫺/⫺ spermatocytes from that of wild-type mice. Spo11 only localized to a subset of synapsed regions, and the only consistent observation was that these regions appeared to be homologously synapsed (Figure 6B). In a mei1 mouse, there was considerably more synapsis, and the fluorescent intensity of Spo11 was just slightly lower than that seen in normal spermatocytes (Figure 6C). There appeared to be full synapsis between homologs (arrow, Figure 6C) as determined by ␣Scp3 staining but there was also some nonhomologous synapsis as well (asterisks, Figure 6C). Morc⫺/⫺ mice undergo very little synapsis, and Spo11 could not be detected in those spermatocytes (data not shown).

Partial Rescue of Meiosis by the DNA Damaging Agent Cisplatin Cisplatin is a chemotherapeutic agent that forms many different adducts with DNA, removal of some of which would require formation of a DNA DSB (Bhattacharyya et al., 2000 and references therein). Previous work showed cisplatin increases the amount of meiotic crossover products by nearly 2-fold (Hanneman et al., 1997). We used cisplatin to induce DSBs in arrested spermatocytes in our Spo11⫺/⫺ mice to see whether they progressed beyond the zygotene-like stage we routinely observed. After cisplatin administration, mice were sacrificed at different time points and analyzed. As shown above, Spo11⫺/⫺ spermatocytes do not have Rad51 and Dmc1 foci (Figures 4D and 4F, respectively). Three days after cisplatin administration, we were able to detect Rad51 (merged with Scp3, Figure 7A) and Dmc1 foci (merged with Scp3, Figure 7B). Approximately 50% of the Rad51 and Dmc1 foci colocalized within nuclei (Figure 7C). We did not see many nuclei with Rad51/Dmc1 foci one day after cisplatin administration (data not shown), and at five days post cisplatin administration the results were much like those seen after three days (data not shown). Seven days after cisplatin administration, there were very few spermatocytes that stained with ␣Scp3 or had Rad51 or Dmc1 foci; in fact, the absolute number of cells isolated from the testis was severely reduced (unpublished observations). The extent of synapsis in cisplatin-treated mice was quantified by counting the number of centromeres in 100 nuclei stained with CREST antisera in cisplatintreated and untreated Spo11⫺/⫺ mice (Figure 7D). There was a significant decrease in nuclei with 40 centromeres (no synapsis) after cisplatin treatment and an increase of cells with evidence of considerable synapsis (31–33 CREST foci). The true extent of synapsis was determined by staining with ␣Scp1 and CREST antisera (Figure 7E). Regions of synapsis usually extended away from centromeres, and cells with fewer staining centromeres (c ⬍ b ⬍ a) had relatively more Scp1. Thus, cisplatin-generated DSBs promote synapsis in Spo11⫺/⫺ spermatocytes. Generally, 30%–50% of nuclei showed considerable synapsis (as determined by ␣Scp1 staining) after cisplatin administration, as compared to 5%–10% in untreated controls. Discussion Spo11 is required for fertility in both male and female mice, and the meiotic arrest we observed is similar to that seen in other mouse knockouts that cause infertility. Spo11⫺/⫺ deficient spermatocytes do not undergo wildtype levels of synapsis, and the synapsis that does occur is mostly between nonhomologs. The phenotype in mouse is more similar to S. cerevisiae and C. cinereus than to C. elegans and Drosophila, where SC formation is essentially unaffected in Spo11 mutants. Thus, large genome size and complexity alone do not imply a need for a Spo11-independent mechanism for chromosome synapsis as seen in C. elegans and Drosophila. Our data on the phenotype of this knockout is in agreement with that in the accompanying study of Baudat and coworkers (Baudat et al. 2000 [this issue of Molecular Cell]). Spo11 was first localized to discrete foci during lepto-

Figure 7. Partial Rescue of Meiotic Arrest in Spo11⫺/⫺ Spermatocytes (A) Induction of Rad51 foci in Spo11⫺/⫺ spermatocytes 3 days after cisplatin administration. Merged image of ␣Scp3 (red) and ␣Rad51 (green). Cells were in zygotene-like stage. The images were initially viewed at 1000⫻ magnification. Four nuclei are shown; the scale bar is 10 ␮m. Overlap of red and green signals results in yellow signal. (B) Induction of Dmc1 foci in Spo11⫺/⫺ spermatocytes 3 days after cisplatin administration. Merged image of ␣Scp3 (red) and ␣Dmc1 (green). Cells were in zygotene-like stage; six nuclei are shown. (C) Colocalization of Rad51 and Dmc1 foci in Spo11⫺/⫺ spermatocyte 3 days after cisplatin administration. Merged image of ␣Rad51 (red) and ␣Dmc1 (green). (D) Graphic representation of increased synapsis seen in Spo11⫺/⫺ spermatocytes after cisplatin administration. One ⫺/⫺ mouse was treated with cisplatin for 3 days, the other was injected with saline. The mice were from the same litter and ⱖ5 weeks old when injected. One hundred nuclei were analyzed for each sample, and the number of CREST signals (centromeres) were recorded. (E) Colocalization of Scp1 and centromeres (CREST antisera) in Spo11⫺/⫺ spermatocytes 3 days after cisplatin administration. Merged image of ␣Scp1 (red) and CREST (green). Three nuclei are shown, (a) has 33 centromeres; (b), 30, and (c), 28. In many instances, synapsis extends away from the centromere. Note more ␣Scp1 staining coincident with fewer centromeres.

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tene, when DSBs are believed to occur, and later only along synapsed regions of homologous chromosomes during zygotene and pachytene, including the PAR of the XY bivalent. The meiotic defect in Spo11⫺/⫺ mice is qualitatively different from other mouse meiotic mutants that presumably function downstream in the recombination process. For example, Atm⫺/⫺ and Dmc1⫺/⫺ spermatocytes also arrest during meiotic prophase I, but Atm plays a more global role in genome surveillance and the effects are pleiotropic, while the meiotic arrest in Dmc1⫺/⫺ mice may be due to a checkpoint activated by unrepaired DSBs. Our Spo11⫺/⫺ mice were not subjected to an intensive analysis of somatic tissues to detect effects other than a disruption of meiosis. Histological analysis of cross sections from thymus, in which there is a high level of Spo11 expression in comparison to other somatic tissues (Romanienko and Camerini-Otero, 1999), did not indicate any obvious alterations in the absence of Spo11 (unpublished observations). The defect seen in fetal ovary (Figures 3C and 3D) can be attributed to the meiotic function of Spo11, but it would be interesting to look earlier in gonad development to see if the premeiotic defect we see in fetal testes (data not shown) can be detected earlier in primordial germ cells. The morphological change we saw in Spo11⫺/⫺ fetal testes may be a reflection of some defect in premeiotic S phase in Spo11⫺/⫺ mice, as was seen in SPO11-deleted yeast (Cha et al., 2000). Spo11 Is Required for Meiotic Chromosome Synapsis Indirect immunofluorescent analysis of meiotic chromosome spreads from Spo11⫺/⫺ male mice showed that axial elements form but synapsis was severely affected (Figure 4B). Almost all the synapsis that occurs is between nonhomologs, as revealed by frequent partner switches. The lack of synapsis might be attributable to the absence of meiotic DSBs, but it is also possible that Spo11 plays a role in earlier homolog pairing (Weiner and Kleckner, 1994) and/or synapsis itself. Normal synapsis in mouse is also dependent on a number of proteins involved in meiotic recombination (Atm, Barlow et al., 1998; Dmc1, Pittman et al., 1998; Yoshida et al., 1998; Msh4, Kneitz et al., 2000; and Msh5, de Vries et al., 1999; Edelmann et al., 1999). Synapsis was also defective in Scp3⫺/⫺ males, but female mice were fertile (Yuan et al., 2000). The dependence of normal synapsis on recombination is central to the deficiency seen in Spo11⫺/⫺ spermatocytes. Either the failure to initiate recombination signals an arrest and normal synapsis does not occur, or the lack of recombination results in a subsequent failure to complete synapsis that triggers the arrest. A mutant mouse that can bypass the requirement for DSB formation and recombination and permits normal synapsis would argue for the second model. Spo11 Localization Is Consistent with Roles in DSB Formation and Synapsis Spo11 was localized in discrete foci during leptotene (Figure 5A) and more intensely at synapsed regions of homologous chromosomes during zygotene and pachy-

tene in wild-type mice (Figures 5B, 5C, and 5E). Spo11 makes DSBs during leptotene in yeast (Roeder, 1997), but the temporal events of meiotic recombination have not been studied in great detail in mouse. The localization of mouse Rad51 to discrete foci during leptotene (Tarsounas et al., 1999) is reasonable evidence that DSBs occur at this stage. Our demonstration that the formation of Rad51 foci in mouse is Spo11-dependent, just as in yeast, is further evidence that DSBs occur during leptotene. Since we were able to detect Rad51 foci in Spo11⫺/⫺ spermatocytes after administration of cisplatin, it is therefore reasonable to conclude that mouse Spo11 generates meiotic DSBs. The discontinuous pattern of Spo11 on pachytene chromosomes (Figures 5C and 5E) is unlike that seen using antibodies against the lateral (Scp3) and central (Scp1) element components of the SC, and it would be interesting to see whether the distribution of Spo11 is specific for each chromosome. That Spo11 is expressed at its highest level in pachytene spermatocytes in mouse (Shannon et al., 1999) and during pachytene in S. cerevisiae (Atcheson et al., 1987; Chu et al., 1998) is consistent with its detection along synapsed regions of paired homologs, but pachytene localization does not necessarily imply a role in pachytene. Mutant Mice Reveal a Spo11 Splicing Defect in Arrested Spermatocytes and Suggest a Role for Spo11 in Stabilizing Homologous Synapsis Analysis of Spo11 localization and expression in several mutant mouse lines allowed us to speculate on the alternate forms of Spo11 and what function they may encode. The RT-PCR results from the mutant mice show a link between meiotic progression from zygotene to pachytene and the removal of exon 2 from the Spo11 transcript (Figure 6A). The predominant form of Spo11 in wild-type adult testes does not contain exon 2. The simplest model suggests that the larger form of Spo11 (Spo11␤) is the catalytic form that generates DSBs, while the smaller form (Spo11␣) plays a structural role in the developing SC. Further evidence for this model is the fact that Rad51 foci (marker for DSBs) form in Dmc1⫺/⫺ spermatocytes (Pittman et al., 1998) (where there is little of the smaller Spo11␣ transcript) with the same kinetics as wild-type mice. mei1 mice show more homologous synapsis than Dmc1 and Atm mutants (data presented above and unpublished observations) and, coincidentally, more splicing to Spo11␣. The finding that DNA ligase III-␤ splicing is unaffected in the mutant mice we analyzed indicates there may be multiple checkpoints regulating different facets of meiotic progression, and that the arrest per se is not responsible for the splicing defect. Our interpretation of immunofluorescence results from Dmc1⫺/⫺ and mei1 spermatocytes is that Spo11 localizes to regions of synapsis between homologs. Very little staining is seen in regions of incorrect synapsis, so one can speculate that Spo11 is required for synapsis between homologs or perhaps stabilizes synapsis between homologs. It is not clear whether Spo11 can discriminate between homologous and nonhomologous synapsis or whether the SC formed between nonhomologs is fundamentally different in a way that is not immediately obvious with our cytological criteria.

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We note, however, that this picture might be an oversimplification. Preliminary results indicate that Brca1Co/Co mice can progress and arrest in pachytene with fully synapsed chromosomes but still do not splice out exon 2 (unpublished observations). Partial Meiotic Rescue in Spo11⫺/⫺ Spermatocytes by Cisplatin Argues for a Role of Spo11 in Generating DSBs We partially rescued the meiotic arrest seen in our Spo11 knockout by generating DNA damage with the chemotherapeutic agent cisplatin. We were able to detect Rad51/Dmc1 foci, the presence of which is good evidence for formation of cisplatin-induced DSBs. We were also able to promote synapsis, indicating that DNA DSBs may influence SC formation, but wild-type levels of synapsis were not observed. In any case, DSBs of any origin, whether generated by Spo11 or cisplatin, and recombination are necessary and sufficient to promote significant levels of synapsis, and the postulated second role of Spo11 may be in stabilizing homologous synapsis. Experimental Procedures Targeted Inactivation of the Mouse Spo11 Gene The targeting construct was based on ploxPneo (Yang et al., 1998) (a gift from Chuxia Deng). A 3.3 kb BspEI/ApaI genomic fragment containing the 5⬘ region of mouse Spo11 was blunted and cloned into the blunted EcoRI site in ploxPneo between the TK gene and PGKneo cassette in the opposite orientation of transcription of the neo gene. This construct was designated pTarget1. A 1.1 kb SmaI/ EcoRV genomic fragment containing intron 1 sequence was cloned into XhoI-cut, blunted pTarget1 on the other side of the neo cassette. This step removed the first 35 nucleotides of intron 1, which contained the splice donor site. This effectively deleted 140 bp of the Spo11 locus and inserted the neo cassette; the construct was designated pTarget2. A 9 kb EcoRI/AatII genomic fragment containing exons 2–9 was blunted and cloned into pTarget2 cut with Hpa I, resulting in the final 20 kb targeting vector, pTarget3. The final cloning step resulted in the deletion of an additional 110 bp from intron 1, including the EcoRI site. PTarget3 was linearized with NotI and prepared for targeting. Ninety micrograms of linearized pTarget3 was electroporated into 1.8 ⫻ 107 TC1 ES cells (Deng et al., 1996) (a gift from Chu-Xia Deng) and seeded onto mitomycin C–treated mouse embryonic feeder cells (Incyte Genomics). Colonies were selected in media containing LIF (GIBCO–BRL), 350 ␮g/ml G418, and 5 ␮M gancyclovir (a gift from Rick Proia). Colonies were picked, expanded, and genomic DNA was extracted for Southern analysis. The removal of the EcoRI site in intron 1 and insertion of the neo cassette made the mutated allele 3 kb (16 kb) larger than the wildtype allele (13 kb). The probe was derived from exons 10–13 of the mouse Spo11 cDNA. One correctly targeted ES clone was injected into C57/B6 blastocysts to obtain germline transmission. Mice obtained after germline transmission were screened by PCR on genomic DNA isolated from tail tips. Primers for PCR genotyping were Spo11-1, 5⬘-TGTCCCGCGGTCAGTGGTGCAG-3⬘ and Spo11-2, 5⬘-TCCAGGGCGTCGAAGAACGAGG-3⬘, which amplified a 150 bp product from genomic DNA. Spo11-2 lies within the deleted region and would not be present in the targeted allele. The neo primers were Neo5⬘-Target, 5⬘-GTACTCGGATGGAAGCCGGTCTT-3⬘ and Neo3⬘Target, 5⬘-GCCAAGCTCTTCAGCAATATCACG-3⬘. The amplified product was 280 bp and was only present if the targeted allele was present. The PCR protocol was 94⬚C, 2 min; 94⬚C, 15 s; and 68⬚C, 45 s for 32 cycles followed by 72⬚C, 10 min with 100 ng of genomic DNA in a 20 ␮l reaction using the Advantage cDNA Polymerase Mix (Clontech). Analysis of Spo11 Knockout Mice Northern blot analysis was done on poly(A)⫹ RNA prepared from testes of the corresponding genotypes using Trizol (GIBCO–BRL)

and the Micro Poly(A) Pure Kit (Ambion). Two micrograms of purified mRNA was run, transferred, and probed sequentially with the mouse Spo11 cDNA and the mouse ␤-actin cDNA (Sigma). Tissues for histological examination were removed and fixed overnight in neutral-buffered 10% formalin (Sigma). Tissue was embedded in paraffin and 6 ␮m sections were cut. Fetal mice were removed from the mother and placed in fixative. The fetal age of the mice was determined by comparison of external facial and body morphology, and their age was placed at 15 dpc (Rugh, 1968). Before fixation, a piece of tail was removed for genotyping. Male mice were confirmed by PCR amplification of the mouse male-specific Sry gene (GenBank accession number U70641) using primers Sry5⬘, 5⬘-GCCCAGCA GAATCCCAGCATGCA-3⬘ and Sry3⬘, 5⬘-TTTTGTTGAGGCAACTG CAGGCTG-3⬘. The PCR product was 250 bp, and the cycling protocol was 94⬚C, 2 min; 94⬚C, 15 s; and 68⬚C, 45 s for 35 cycles followed by 72⬚C, 10 min. Sections were stained with hematoxylin and eosin (H&E) or analyzed for apoptosis using the TACS 2 TdT-In situ Apoptosis Detection Kit (Trevigen). Antibodies for Immunofluorescence An antibody was raised against a peptide, MmPep-1 (amino acids 224–242, EKDATFQRLLDDNFCSRMS, encoded in exon 8 of mouse Spo11 [Romanienko and Camerini-Otero, 1999]), that was synthesized on MAP (Multi-Antigenic Peptide) resin (Novabiochem) on a PerSeptive Biosystems peptide synthesizer. The crude synthesis product was used to generate polyclonal antisera in two rabbits (Covance). Antisera was checked by Western blot analysis of mouse Spo11:GFP fusion protein expressed in mouse 3T3 cells (ATCC). ␣-Spo11 was affinity purified by passage over a column to which was coupled HPLC-purified MmPep-1. Peptide coupling was done using AminoLink Coupling Gel (Pierce), and binding and elution were done with ImmunoPure Gentle Binding buffer and ImmunoPure Gentle Elution buffer, respectively (Pierce). The purified antiserum was dialyzed into Tris-buffered saline, .05% sodium azide, and concentrated to approximately 1mg/ml using Slide-A-Lyzer Concentrating Solution (Pierce). The purified antibody was aliquoted and frozen at ⫺20⬚C. Primary antibodies (in addition to rabbit ␣-mouse Spo11 at 1:10 or 1:20) used for immunofluorescence were mouse ␣-rat SCP1 at 1:10 (a gift from Christa Heyting, Wageningen University, The Netherlands), mouse ␣-hamster SCP3 (Cor1) at 1:1000 (a gift from Peter Moens, York University, Toronto, Canada), CREST human antisera at 1:200 (a gift from Bill Brinkley, Baylor College, Texas), goat ␣-Dmc1 at 1:200 (C-20) (Santa Cruz Biotechnology), and rabbit ␣-Rad51 at 1:200 (H-92) (Santa Cruz Biotechnology). All secondary antibodies (Jackson Immunoresearch Laboratories) were used at 1:200. Secondary antibodies were either conjugated with Rhodamine Red-X or FITC and were generated in goats or donkeys. Meiotic Chromosome Spread Preparation and Immunofluorescence Chromosome spreads were prepared as follows: testes were dissected, seminiferous tubules were digested with .5 mg/ml collagenase in RPMI 1640 high glucose media supplemented with amino acids (GIBCO–BRL) for 20 min at 33⬚C with gentle shaking, digested with .25 mg/ml trypsin, 1 ␮g/ml DNase I for 20 min at 33⬚C with gentle shaking, then filtered through a 40 ␮m cell strainer (Falcon). Soybean trypsin inhibitor, SBTI (GIBCO–BRL), was added at 100 ␮g/ml, and the cell suspension was centrifuged at 800 ⫻ g for 5 min, washed with media containing SBTI, and recentrifuged. The supernatant was removed, and cells were resuspended in .5% NaCl along with any residual media that remained after aspiration. The cell suspension was pipetted onto glass slides with hydrophobic rings (Becton Dickinson), and the cells were allowed to swell. When the cells began to adhere to the slide surface, the slides were fixed in 2% paraformaldehyde, .03% SDS for 3 min, then in 2% paraformaldehyde for 3 min. Slides were washed 3⫻ in .4% Photo-Flo 200 (Kodak) and allowed to air dry. Slides were washed 2⫻ in PBS, blocking solution (10% serum from goat or donkey, 3% BSA, .05% Triton X-100 in PBS) was added to the hydrophobic rings, and slides were incubated for 2 hr in a humid chamber at room temperature. Primary antibodies were diluted in blocking buffer, and slides were incubated in a humid cham-

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ber at 37⬚C for 1 hr. After washing, secondary antibodies were added for 20 min at room temperature. The slides were washed and allowed to dry. Vectashield Mounting Medium with DAPI (Vector Laboratories) was added and the slides were viewed. The chromosome spread and antibody incubation procedure was adapted from a protocol from Peter Moens. Slides were examined with a Leica fluorescent microscope, and individual FITC and rhodamine images were captured as eight bit color images at 1000⫻ magnification using an oil immersion lens. The images were processed using Adobe Photoshop 4.0 and merged. Balb/c mice were used for localization of Spo11. Juvenile male mice of ages 10–21 days postpartum (dpp) were used to prepare the spreads. The mutant mice used in this study are as follows, Atm⫺/⫺ (Barlow et al., 1996) (purchased from Jackson Laboratories), Dmc1⫺/⫺ (Pittman et al., 1998) and mei1 (Munroe et al., 2000) (a gift from John Schimenti, Jackson Laboratories, Bar Harbor, Maine), and Morc⫺/⫺ (Watson et al., 1998) (a gift from Andrew Zinn and Mark Watson, University of Texas-Southwestern, Dallas, Texas). The mutant mice were ⱖ5 weeks of age at time of assaying. Comparison of Spo11 Transcripts in Mutant and Wild-Type Mice by RT-PCR The primers used to amplify mouse Spo11 were 5⬘MmSpo, 5⬘-CTG TTGGCCATGGTGAGAGAGG-3⬘ and MmSpoSeq1, 5⬘-TCCTTGAA TGTTAGTCGGCACAGC-3⬘. The product with exon 2 is 560 bp and without exon 2 is 460 bp. The PCR protocol was 94⬚C, 2 min; 94⬚C, 15 s; and 68⬚C, 1 min for 33 cycles followed by 72⬚C, 10 min. The Aop2 control PCR and primers were as described (Pittman et al., 1998). Poly(A)⫹ RNA was isolated from mouse testis tissue as described earlier, and cDNA synthesis was done as described (Romanienko and Camerini-Otero, 1999). Cisplatin Administration Cisplatin (Sigma) was administered as described (Hanneman et al., 1997). Mice were sacrificed at time points indicated, and testes were removed for meiotic chromosome spread preparation and/or histological examination. CREST foci were counted in a methodical manner with no bias in nuclei selection other than the omission of leptotene spermatocytes, which are apparently normal in Spo11⫺/⫺ mice. All cells were in a zygotene-like stage. Foci that were partially overlapped such that there was no space between them were counted as one focus. Adminsitration of cisplatin was performed according to ACUC guidelines at the NIH. Acknowledgments We are grateful to Chu-Xia Deng and Cuiling Li for blastocyst injection, generating chimeric mice, and providing ES cells; Rick Proia and his lab for the gancyclovir and targeting advice; April Anderson for technical assistance; Linda Robinson for her assistance; and George Poy for oligo and peptide synthesis. We would like to thank those who graciously provided the mutant mice, John Schimenti for the Dmc1⫺/⫺ and mei mice, and Mark Watson and Andrew Zinn for the Morc⫺/⫺ mice. We are especially grateful to Bill Brinkley for the ␣CREST antisera, Christa Heyting for the ␣SCP1 antibody, and Peter Moens for the ␣SCP3 antisera and advice on performing meiotic spreads. Finally, we want to thank Michael Lichten and Rick Proia for their insightful comments on the manuscript and Chu-Xia Deng and Kim McKim for communicating unpublished results. Received August 4, 2000; revised September 19, 2000. References Aravind, L., Leipe, D.D., and Koonin, E.V. (1998). Toprim- a conserved catalytic domain in type IA and II topoisomerases, DnaGtype primases, OLD family nucleases and RecR proteins. Nucleic Acids Res. 26, 4205–4213. Atcheson, C.L., DiDomenico, B., Frackman, S., Esposito, R.E., and Elder, R.T. (1987). Isolation, DNA sequence, and regulation of a meiosis-specific eukaryotic recombination gene. Proc. Natl. Acad. Sci. USA 84, 8035–8039.

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