Synaptonemal complex extension from clustered PNAS PLUS telomeres mediates full-length pairing in Schmidtea mediterranea

Youbin Xianga, Danny E. Millera,b, Eric J. Rossa,c, Alejandro Sánchez Alvaradoa,c, and R. Scott Hawleya,b,1

aStowers Institute for Medical Research, Kansas City, MO 64110; bDepartment of Molecular and Integrative Physiology, Kansas University Medical Center, Kansas City, KS 66160; and cHoward Hughes Medical Institute, Stowers Institute for Medical Research, Kansas City, MO 64110

Contributed by R. Scott Hawley, October 24, 2014 (sent for review August 12, 2014) In the 1920s, József Gelei proposed that chromosome pairing in served in many, if not most, other organisms (3, 4). The telomere flatworms resulted from the formation of a telomere bouquet bouquet is a structure formed when telomeres cluster at a small followed by the extension of synapsis from telomeres at the base area of the nuclear periphery (5). The precise role of this bouquet of the bouquet, thus facilitating homolog pairing in a processive in mediating pairing and/or synapsis remains unclear (for review, manner. A modern interpretation of Gelei’s model postulates that see ref. 6). the synaptonemal complex (SC) is nucleated close to the telomeres In contrast, DSBs are not required for the formation of SC in and then extends progressively along the full length of chromo- Drosophila females (7), and a telomere-driven meiotic bouquet is some arms. We used the easily visible meiotic , not observed (8, 9). Although the formation of the SC at clustered a well-characterized genome, and RNAi in the sexual biotype of the centromeres and at a small number of interstitial sites is concurrent Schmidtea mediterranea planarian to test that hypothesis. By identi- with the establishment of chromosome pairing (9, 10), the absence S. mediterranea fying and characterizing homologs of encoding of the SC prevents meiotic pairing both at the centromeres and synaptonemal complex 1 (SYCP1), the topoisomerase-like along the arms of homologous chromosomes (11–13). Moreover, protein SPO11, and RAD51, a key player in homologous recombi- SC formation is required for normal levels of DSB formation (14, nation, we confirmed that SC formation begins near the telo- 15) as well as for the maturation of recombination intermediates meres and progresses along chromosome arms during zygotene. into crossovers. Similarly, in Caenorhabditis elegans, DSBs are not Although distal regions pair at the time of bouquet formation, required for synapsis (16). Rather, pairing is initiated at telomere- pairing of a unique interstitial locus is not observed until the for- mation of full-length SC at pachytene. Moreover, neither full ex- adjacent pairing centers (PCs) on each homolog pair, and the pro- tension of the SC nor homologous pairing is dependent on the cessive extension of SC from the PCs is required to establish the formation of double-strand breaks. These findings validate Gelei’s alignment of homologous chromosomes along their full lengths (17, speculation that full-length pairing of homologous chromosomes 18). As opposed to its much later role(s) in mammals and yeast, the is mediated by the extension of the SC formed near the telomeres. SC in these systems appears to be critical for establishing tight pairing S. mediterranea thus becomes the first organism described (to our and maintaining tight homolog associations, thus providing an envi- knowledge) that forms a canonical telomere bouquet but does not ronment in which DSBs can mature into functional crossovers. require double-strand breaks for synapsis between homologous The large differences in meiosis observedinafewwell-charac- chromosomes. However, the initiation of SC formation at the base terized model systems suggest that much more diversity may exist of the telomere bouquet, which then is followed by full-length ho- in nature. Therefore it seems prudent to begin an exploration of mologous pairing in planarian spermatocytes, is not observed in meiosis in previously uncharacterized classes of organisms. In the other species and may not be conserved. Significance meiosis | synaptonemal complex | telomere bouquet | planarian | homologous chromosome pairing In this study we validate a nearly century-old model for chromo- some pairing in flatworms and provide a molecular description of meiotic prophase in flatworms. Specifically, we validate József Genetics functions best as a comparative science. –Kenneth W. Cooper Gelei’s proposal that chromosome pairing in flatworms results from the formation of a telomere bouquet followed by the extension of synapsis from the base of the bouquet, thus facilitating homolog GENETICS lthough most textbooks present meiosis as an almost pairing in a processive manner. This study further advances the Amonolithic process, there is enormous diversity in the mech- groundwork necessary to establish Schmidtea mediterranea as anisms by which the goals of meiosis are achieved. Indeed, the a powerful new meiotic system. The genes identified and the plasticity of the meiotic process is visualized most easily by com- RNAi constructs and antibodies generated during this work help paring the events of meiotic prophase in different organisms. In make planarian meiosis a highly tractable model system. budding yeast, mammalian, and plant systems, initial pairing and alignment steps are followed by the induction of a large number of Author contributions: Y.X. and R.S.H. designed research; Y.X. performed research; Y.X., programmed double-strand breaks (DSBs) (reviewed in ref. 1). The E.J.R., A.S.A., and R.S.H. contributed new reagents/analytic tools; Y.X., D.E.M., E.J.R., and R.S.H. analyzed data; and Y.X., D.E.M., A.S.A., and R.S.H. wrote the paper. formation of DSBs and their maturation into recombination inter- mediates allow the subsequent assembly of a proteinaceous struc- The authors declare no conflict of interest. ture called the synaptonemal complex (SC) along the length of the Freely available online through the PNAS open access option. paired homologs. In addition to other functions, the SC then Data deposition: The sequences reported in this paper have been deposited with the National Center for Biotechnology Information, www.ncbi.nlm.nih.gov (KM487298, facilitates the maturation of a fraction of recombination inter- KM487299,andKM487300) and the SmedSxl_20141024 transcriptome database mediates into mature crossovers, called chiasmata, that eventually generated in this paper is publicly available at smedgd.stowers.org. ensure segregation of chromosomes in meiosis I (2). Budding yeast 1To whom correspondence should be addressed. Email: [email protected]. and mammalian meiosis also are characterized by the presence of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. telomeric bouquets during meiotic prophase, a phenomenon ob- 1073/pnas.1420287111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1420287111 PNAS | Published online November 17, 2014 | E5159–E5168 Downloaded by guest on September 28, 2021 early 20th century, a cytogeneticist named József Gelei proposed SYCP1 orthologs in S. mediterranea, we used mouse SYCP1 [Na- that chromosome pairing in flatworms resulted from the formation tional Center for Biotechnology Information (NCBI) accession no. of a telomere bouquet followed by the extension of synapsis NP_035646] to search a planarian transcriptome database from the base of the bouquet, thus facilitating homolog pairing (the SmedSxl_20141024 database available at smedgd.stowers. in a processive manner (5, 19, 20). A modern interpretation of org) assembled from adult sexual S. mediterranea using tBLASTn Gelei’s model would propose that SC formation is nucleated ini- (25). We identified a transcript with a 1,205-aa ORF that con- tially in the vicinity of the telomeres, perhaps by sites or regions tained a 92-aa peptide fragment (SMED-SYCP1 274-365, NCBI analogous to the PCs observed in C. elegans. The progressive ex- accession no. KM487299) with 35% identity and 52% similarity tension of the SC along chromosome arms then would mediate to mouse SYCP1 (E value 2e-6). Analysis of the protein structure homolog pairing. We set out to test this hypothesis directly in the showed that the 1,205-aa protein has typical TF characteristics, planarian Schmidtea mediterranea. containing both coiled-coil and globular domains, supporting the idea We confirmed Gelei’s observation that synapsis, as assayed by that this protein is SMED-SYCP1 (Fig. S2). Additionally, two pro- SC formation, does indeed begin in the vicinity of the telomeres posedconservedmotifs,CM1andCM2,havebeenidentifiedinthe and then progresses along the chromosome arms during zygo- TF of several organisms (26). To support the identification tene. Although distal regions appear to pair at the time of of SMED-SYCP1 further, we used ClustalX to align SMED-SYCP1 bouquet formation, as assayed by FISH, the pairing of a unique to the TF sequences of several organisms and found that it shares interstitial chromosomal locus is not observed until pachytene. sequence similarity with CM1 (Fig. S3). We further show that, although full-length pairing requires SC To confirm that this protein is an SC component in planarians, we formation, neither SC formation nor homolog pairing requires raised an antibody against SYCP1 that distinctly and specifically the formation of DSBs. S. mediterranea thus becomes (to our stained spermatocytes and oocytes (Fig. S4). Staging of prophase in knowledge) the first organism to be identified that forms a ca- spermatocytes was assessed using chromosome morphology, as de- nonical telomere bouquet but does not require DSBs to allow scribed by Chretien (23). Immunostaining with the anti-SYCP1 anti- synapsis between homologous chromosomes. Rather, our data body showed little to no SYCP1 staining in leptotene nuclei (Fig. 1A). validate Gelei’s hypothesis that the full-length pairing of ho- mologous chromosomes is mediated by the extension of SC that is formed initially near the telomeres. DAPI SYCP1 Merge sycp1(RNAi) Results DAPI AESYCP1 To present our tests of Gelei’s hypothesis, we first must describe the biology of meiotic prophase in the male germ line of this

organism and present both our identification of the synaptone- Leptotene mal complex protein 1 (SYCP1)-encoding and the de- velopment of a FISH-based method to assess chromosome B pairing. We then describe the isolation of the topoisomerase-like protein SPO11 and RAD51 (a key player in homologous re- combination) homologs in S. mediterranea. Early zygotene The Basic Biology of Meiosis in S. mediterranea. The planarian S. mediterranea is a diploid species of freshwater flatworm with C four chromosome pairs (2N = 8) (Fig. S1). S. mediterranea exists in both sexual and asexual forms. Asexual planarians reproduce

through transverse fission of the body, whereas sexual planarians Late zygotene are obligate cross-fertilizing hermaphrodites. Sexual animals, which DAPI SYCP1 Merge are the focus of our investigations, have one pair of ventrally located D ovaries behind the cephalic ganglia and numerous testes located dorsolaterally along the length of both sides of the body (21). Planarian testes possess a testis lobe, a structure similar to seminiferous tubules in mammals, in which spermatogenesis Pachytene 1μm 1μm 1μm proceeds (22). As described by Chretien (23), meiotic sperma- F DAPI SYCP1 Merge tocytes are located within the testis lobe, which is well defined and easily distinguished from surrounding somatic cells. Pre- meiotic cells undergo mitotic cell division at the peripheral portion of the lobe, and spermatocytes migrate toward the lumen

as they mature into spermatids and spermatozoa. Chretien (23) Zygotene primarily used nuclear morphology to stage meiotic prophase in spermatocytes, but now, as we show below, we can resolve meiotic progression in greater detail based on SC morphology, allowing us sycp1(RNAi) to differentiate between leptotene, zygotene, and pachytene stages. Upon completion of pachytene, chromosomes enter a diffuse stage in which morphology becomes difficult to assess. Pachytene-like – Identification of SMED-SYCP1, a Component of the Planarian SC. Fig. 1. Localization of SYCP1. (A D) Representative wild-type leptotene (A), Synapsis during meiotic prophase I is an important event that early zygotene (B), late zygotene (C), and pachytene (D) spermatocyte nuclei facilitates proper homologous chromosome pairing, recombination, stained with DAPI and antibodies to SYCP1. Arrowheads in D indicate SYCP1 staining between parallel DAPI tracks, and boxed areas are enlarged at right. and segregation. The SC is an essential component of this process (E) Immunostaining with antibodies against SYCP1 in an sycp1(RNAi) testis (13). Among the most important proteins of the SC are transverse shows no SYCP1 stretches. (F) Representative zygotene and pachytene nuclei filament (TF) proteins. In many organisms, the TF appears to be in sycp1(RNAi).InA–D and F, DAPI panels are single z-slices; SYCP1 panels are composed of a single protein (SYCP1 in mammals) (24). To find projections. (Scale bars, 5 μm unless otherwise indicated.)

E5160 | www.pnas.org/cgi/doi/10.1073/pnas.1420287111 Xiang et al. Downloaded by guest on September 28, 2021 In zygotene spermatocytes, short stretches of SYCP1 staining could be (n = 64) of foci were <1.5 μm apart, indicating that homologs PNAS PLUS observed at a small area near the nuclear periphery in some cells (Fig. had paired by this stage (Fig. 3 B and D). These results were 1B) and covering approximately half of the nucleus in other cells surprising, because substantial homolog pairing precedes syn- (Fig. 1C). These figures represent early zygotene and late zy- apsis in budding yeast (30–32) and mammals (33, 34). gotene nuclei, respectively. The SYCP1 stretches became pro- We also identified and analyzed a distal euchromatic probe that gressively longer as nuclei progressed through meiosis, and in localizes near the telomere of one arm of chromosome II (ChrII) pachytene nuclei the stretches followed parallel DAPI-staining (Fig. S6A). We found that in wild-type spermatocyte nuclei, tracks, reached full length, and filled the whole nucleus (Fig. chromosome ends were not yet paired in leptotene, but 96.5% 1D). These images provide strong evidence that our proposed (n = 57) had paired by zygotene and remained paired through SYCP1 homolog is indeed a functional SC protein. Thus, the pachytene (100%, n = 46) (Fig. S6 B and C). The timing of distal staging of meiotic prophase in spermatocytes may be accom- versus interstitial pairing is consistent with Gelei’s hypothesis that plished either by following SYCP1 localization or by examining full-length pairing is driven progressively by synapsis. DNA morphology. We next examined whether the SC protein SYCP1 is required for To verify the specificity of the antibody, we used RNAi knock- the observed pairing of the medial ChrIII probe at pachytene. At down to examine several aspects of meiotic progression in sycp1 zygotene, knockdown of sycp1 by RNAi had little effect on the (RNAi) animals.AsshowninFig.1E, RNAi knockdown of sycp1 distribution of the distances between ChrIII probe foci, with 98% both ablated the immunofluorescent signal of the anti-SYCP1 an- (n = 62) of foci not paired (Fig. 3 A and C). By pachytene-like tibody and disrupted meiotic progression in spermatocytes. Al- stages, only 24.6% (n = 61) of sycp1(RNAi) nuclei were paired, though we were able to distinguish normal-looking zygotene nuclei compared with 93.8% in wild-type nuclei (Fig. 3 B and D), in- basedonDAPIstaininginsycp1(RNAi), we did not observe the dicating that pairing of the region marked by the ChrIII probe is parallel DNA tracks that normally are evident in pachytene nuclei greatly reduced in sycp1(RNAi) spermatocytes. However, knock- (Fig. 1 D and F). Chromosomes appeared to be less compact in down of sycp1 by RNAi showed no obvious defect in the sub- sycp1(RNAi) pachytene nuclei than in zygotene nuclei, as assayed by terminal ChrII probe with respect to the pairing of this DAPI staining, and the nuclei appeared to have a more irregular chromosome end (Fig. S6D). The distribution of the distances surface. Based on these observations, we defined these nuclei as between ChrII probe foci in sycp1(RNAi) animals indicated that “pachytene-like.” Some gamete formation still could be observed, 94.5% (n = 55) of foci were paired in zygotene (Fig. S6 B and D) but the gametes either formed abnormal spermatids with defective and that 100% (n = 37) of foci were paired by pachytene-like flagella or stopped their developmental progression altogether, stages. Thus SC formation is not required to maintain the pairing presumably leading to cell death. of distal (telomere-adjacent) sites; rather, such associations appear to be created and maintained by telomere clustering. SYCP1 Localization Begins at the Base of Telomere Bouquets of We conclude from these experiments that SYCP1, and thus SC Meiotic Prophase Nuclei. In many organisms, such as yeast and extension, is required for full-length homologous chromosome mice, telomeres cluster into a bouquet early in prophase (5, 19). pairing at pachytene. In this sense, the events of meiotic prophase Gelei (20, 27) demonstrated that telomere bouquets also are in planarian meiosis may be similar to those seen in C. elegans a prominent feature of flatworm meiosis, an observation con- firmed in S. mediterranea by Chretien (23). To follow the bou- quet in this study, we used recent findings by Tan et al. (28), who showed that S. mediterranea has the same telomere repeat A Leptotene Zygotene Pachytene unit, (TTAGGG)n, found in vertebrates. A FISH probe to the (TTAGGG)n repeat revealed that telomeres were clustered near the nuclear periphery in the majority of zygotene (90.2%, n = 51) and pachytene (94.2%, n = 69) nuclei (Fig. 2A). The bouquet persisted at least until the end of pachytene, as indicated by SC disassembly (Fig. S5). Interestingly, we observed that the small area in which SYCP1 stretches first could be detected in early zygotene always localized at the nuclear periphery near the base of B the telomere bouquet (Fig. 2B). In addition, knockdown of sycp1 Telomeres DAPI SYCP1 DAPI Merge did not affect telomere clustering (Fig. 3 A and B). This result shows that, as suggested initially almost a century ago, synapsis in planarians likely initiates at or near the bouquet base (20, 27). GENETICS

Full-Length Homologous Pairing in S. mediterranea Occurs Progressively Early zygotene and Requires SC Extension. Pairingisanintegralstepinmeiotic progression. To assess the timing of meiotic pairing, we used a method similar to oligopaints (29)—but on a smaller scale—to identify a euchromatic probe that localizes to the middle of the arm

on chromosome III (ChrIII) (Fig. S1). In wild-type spermatocytes, Late zygotene we expect to observe two well-separated ChrIII foci on spatially separated homologous chromosomes and either one focus or two closely associated foci on chromosomes that have paired. We also would expect chromosomes to be paired in pachytene. Using FISH

analysis, we found that in wild-type spermatocytes, the majority Pachytene (93.8%, n = 64) of ChrIII foci in pachytene nuclei were within 1.5 μm of each other; thus we consider two foci ≤1.5 μmapartto Fig. 2. SYCP1 localization initiates at the base of the telomere bouquet. (A) be paired and two foci >1.5 μm apart to be unpaired. = A FISH probe to the (TTAGGG)n telomere repeat shows telomere dynamics at In 88.2% (n 68) of zygotene nuclei, the distance between different prophase I stages of meiosis in spermatocyte nuclei. (B) Immu- ChrIII probe foci was >1.5 μm, suggesting that homologs had not nostaining indicates that SYCP1 localization initiates near the base of the yet paired at this locus (Fig. 3 A and C). By pachytene, 93.8% telomere bouquet. (Scale bars, 5 μm.)

Xiang et al. PNAS | Published online November 17, 2014 | E5161 Downloaded by guest on September 28, 2021 DAPI Telomeres Chr III probe Merge mRNA of spo11 is transspliced. Analysis of the resulting se- A quence indicates that Smed-spo11 encodes a 300-aa protein containing six of the seven highly conserved regions character- istic of SPO11 orthologs (Fig. S7) (38).

Wild type To study the timing and spatial control of DSB formation in planarian meiosis, we needed to identify a protein that marks the sites of DSB formation. After DSBs form, the conserved DNA- Zygotene binding protein RAD51 binds to resected DSBs along with other DNA repair proteins to facilitate the creation of recombination/

sycp1(RNAi) repair intermediates (39), and antibodies against RAD51 com- B monly are used to identify DSB sites in a number of organisms (1). Although Chretien (23) previously had identified a potential homolog of rad51, the attempts to generate an antibody against

Wild type the protein were unsuccessful. Our independent search of SmedGD for homologs of Mus musculus rad51 (NCBI accession no. NP_035364) identified two candidates, mk4.015647.00.01 (E value 1e-143) and mk4.016937.01.01 (E value 7e-75). A tBLASTn search of SmedGD using the two DNA sequences further confirmed that mk4.015647.00.01 (NCBI accession no. KM487300)—the gene sycp1(RNAi)

Pachytene/Pachytene-like identified by Chretien (23)—was highly homologous to rad51. We expressed this protein in E. coli for antibody generation. C Zygotene D Pachytene/Pachytene-like We used the localization of RAD51 in meiotic nuclei to visu- Wild type (n=68) sycp1(RNAi) (n=62) Wild type (n=64) sycp1(RNAi) (n=61) 25 70 alize sites of DSB formation (Fig. 4). Immunostaining in wild-type

20 60 spermatocytes showed that RAD51 foci were observed first in 50 clusters in early zygotene nuclei that exhibited punctate SYCP1 15 40 localization (Fig. 4A), with an average ± SD of 10.5 ± 5.0 foci per 10 30 = Percent Percent nucleus (n 43) (Fig. 4E). The number of RAD51 foci was 20 5 highest in late zygotene nuclei, which had SYCP1 stretches cov- 10 ± 0 0 ering about half the nucleus, with an average of 16.9 6.4 RAD51 foci per nucleus (n = 89) (Fig. 4 B and E). When nuclei reached 0.5-1.0 0.5-1.0

>1.0-1.5 >1.5-2.0 >2.0-2.5 >2.5-3.0 >3.0-3.5 >3.5-4.0 >4.0-4.5 >4.5-5.0 >5.0-5.5 >1.0-1.5 >1.5-2.0 >2.0-2.5 >2.5-3.0 >3.0-3.5 >3.5-4.0 >4.0-4.5 >4.5-5.0 >5.0-5.5 pachytene, and SYCP1 stretches filled the nucleus, the number of Distance between Distance between RAD51 foci decreased to ∼10.3 ± 2.9 foci per nucleus (n = 31) Chr III probe foci (μm) Chr III probe foci (μm) (Fig. 4 C and E). Although RAD51 foci remained restricted to Fig. 3. SYCP1 is required for homologous chromosome pairing. (A and B) a small area through pachytene in the majority of nuclei, we also = FISH with the (TTAGGG)n telomere repeat probe and the ChrIII probe shows observed a subset of pachytene nuclei (26.8%, n 97) exhibiting bouquet formation and homologous pairing in zygotene and pachytene fewer and more dispersed RAD51 foci (Fig. 4D). We speculate nuclei in both wild type and sycp1(RNAi). DAPI panels are single z-slices; that these may represent nuclei in late pachytene. Depletion of telomere and ChrIII probe panels are projections. (Scale bars, 5 μm.) (C and D) SPO11 by RNAi resulted in an almost complete elimination of The distribution of distances between ChrIII foci in wild type and sycp1(RNAi) RAD51 foci (Fig. 4 E–G). in zygotene and pachytene indicates that SYCP1 is required for pairing. In wild-type nuclei, clusters of RAD51 foci often were observed at or near the nuclear periphery in early zygotene and pachytene – (18), in which the pairing of the majority of the chromosome is (Fig. 4 A C). Immuno-FISH revealed that these RAD51 clusters were located in the vicinity of the telomeres at the base of the mediated by the extension of the SC formed at pairing centers – located near the telomeres. telomere bouquet in zygotene and pachytene (Fig. 4 I J), sug- gesting that DSB formation preferentially occurs at the region Meiotic DSBs Occur Primarily in the Vicinity of the Telomere. Al- located at or near the sites where SC formation begins (Fig. 4A). though the data presented above clearly show that synapsis is However, in the subset of pachytene nuclei with dispersed RAD51 required for full-length pairing in S. mediterranea, they do not foci, the foci displayed no obvious association with the telomeres rule out a role for programmed DSBs in mediating one or both (Fig. 4J). Of course, we cannot know whether these RAD51 foci, of these processes. To address this possibility, we needed to which are localized to interstitial sites, arose from the early round(s) of DSB formation that localized tightly to telomeric regions or confirm that planarians catalyze programmed DSBs using the occurred later in meiotic prophase (Discussion). highly conserved SPO11 topoisomerase (35) and to generate To determine whether the restriction of RAD51 foci to sites at tools to visualize DSB sites. First, we identified the SPO11 or near the bouquet base during early meiotic prophase is the ortholog using the amino acid sequence of mouse SPO11 (NCBI result of a regulated biological process that localizes DSBs to this accession no. AAF87090) to perform a tBLASTn search of region of the nucleus, we irradiated planarians and analyzed the the S. mediterranea genomedatabase(SmedGD,availableat distribution of RAD51 foci during prophase. Immunostaining smedgd.stowers.org) (36). The BLAST search identified a 93-bp showed abundant RAD51 foci distributed throughout the nuclei DNA fragment encoding 31 amino acids highly homologous to at all meiotic stages in spermatocytes (Fig. 4K). These observa- – amino acids 180 210 of mouse SPO11 isoform b (E value 8e-5, tions confirm the specificity of the RAD51 antibody to DSB sites 65% identity) and to portions of other SPO11 proteins from and also suggest that the clustering of RAD51 foci in early multiple organisms. Based on the 93-bp sequence obtained from prophase likely reflects a bias in favor of programmed DSB ′ ′ the BLAST search, the 5 - and 3 -terminal ends of Smed-spo11 formation near telomeres at the onset of synapsis. were amplified by PCR to obtain the full-length spo11 gene (NCBI accession no. KM487298). Because the 5′ end of spo11 DSB Formation Is Not Required for Telomere Bouquet Formation, can be amplified using a primer based on the planarian spliced- Homologous Chromosome Pairing, or Synapsis. spo11(RNAi) ani- leader (SL) sequence (37), and no such SL sequence is present in mals displayed normal meiotic cell progression to form gametes, the vicinity of the first exon of the spo11 gene, we infer that the spermatids, and sperm. To gauge bouquet formation in spo11

E5162 | www.pnas.org/cgi/doi/10.1073/pnas.1420287111 Xiang et al. Downloaded by guest on September 28, 2021 SYCP1 RAD51 Merge E 25 PNAS PLUS A Wild type 20 spo11(RNAi)

15 10

Early zygotene 5

Number of RAD51 foci 0 B Early zygotene Late zygotene Pachytene SYCP1 RAD51 Merge F Late zygotene C Wild type Pachytene Zygotene D spo11(RNAi) G Pachytene

Telomeres RAD51 Merge

H Wild type Pachytene Zygotene

I spo11(RNAi)

K DAPI

Pachytene RAD51 J Pachytene

Fig. 4. Initiation of recombination in wild-type and spo11(RNAi) spermatocytes. (A–D) Projection images of wild-type spermatocytes stained with antibodies

to SYCP1 and RAD51. (A) At early zygotene, RAD51 foci localize to the bouquet base near SYCP1. (B) At late zygotene, SYCP1 stretches extend to half or two- GENETICS thirds of the nucleus, and the number of RAD51 foci peaks. (C) At pachytene, DAPI tracks are clearly formed, SYCP1 extends through the whole nucleus, and the number of RAD51 foci are reduced but still are clustered primarily at or near the bouquet base (see I). (D) In 26.8% of pachytene nuclei (n = 97), RAD51 foci are more dispersed. (E) Quantification of the number of RAD51 foci in early zygotene, late zygotene, and pachytene nuclei from wild-type (n = 163 nuclei in 14 testis sections) and spo11(RNAi) (n = 150 nuclei in 11 testis sections). Comparison of the number of RAD51 foci in wild type and spo11(RNAi) indicates that SPO11 is required for DSB formation. (F and G) Projection images of representative wild-type and spo11(RNAi) nuclei show localization of SYCP1 and

RAD51 foci in zygotene (F) and pachytene (G). (H–J) Projection images of wild-type nuclei stained with DAPI, antibodies to RAD51, and the (TTAGGG)n telomere repeat probe. RAD51 foci cluster near the telomere bouquet base in zygotene (H) and pachytene (I) but are dispersed and unassociated with the telomeres in the 26/97 pachytene nuclei with dispersed RAD51 foci (J). (K) X-ray irradiation produced widespread (nonclustered) RAD51 foci in spermatocyte nuclei. (Scale bars, 5 μm.)

(RNAi) animals, we used the (TTAGGG)n telomere repeat (Fig. 5C), similar to the percentage of unpaired chromosomes probe. Our results showed that knockdown of spo11 did not af- (88.2%. n = 68) in wild-type animals. At pachytene, FISH fect telomere bouquet formation (Fig. 5 A and B). We then used analysis showed that the average distance between foci in RNAi- the euchromatic ChrIII probe to evaluate homologous chromo- treated animals was statistically indistinguishable from that in some pairing in the spo11(RNAi) animals. At zygotene, 93.8% wild-type animals (1.11 ± 0.51 μm vs. 1.00 ± 0.35 μm in wild type; (n = 65) of spo11(RNAi) animals displayed unpaired chromo- two-tailed P value = 0.2135), and the distribution of the distances somes based on the distribution of the distances between foci between foci was very similar between spo11(RNAi) (88.0% of

Xiang et al. PNAS | Published online November 17, 2014 | E5163 Downloaded by guest on September 28, 2021 DAPI Telomeres Chr III probe Merge foci per nucleus (n = 32)versus10.3± 2.9 foci (n = 31) in wild A type (P < 0.00001) (Fig. 6 B and C). That these RAD51 foci do indeed reflect the accumulation of unrepaired Spo11-induced DSBs is demonstrated by the observation that sycp1(RNAi);

Wild type spo11(RNAi) double-knockdown spermatocytes showed a dramati- cally reduced number of RAD51 foci (Fig. 6), similar to the levels Zygotene seen in spo11(RNAi) alone. These data indicate that in planarians, as in mammals, SYCP1 is dispensable for meiotic DSB formation. spo11(RNAi) B DAPI RAD51 Merge A Wild type Wild type Pachytene spo11(RNAi)

C Zygotene D Pachytene

Wild type (n=68) spo11(RNAi) (n=65) Wild type (n=64) spo11(RNAi) (n=58) Zygotene

25 70 sycp1(RNAi)

20 60 50 15 40 10 30 Percent Percent 20 5 10 0 0 sycp1+spo11(RNAi)

0.5-1.0 0.5-1.0 B >1.0-1.5 >1.5-2.0 >2.0-2.5 >2.5-3.0 >3.0-3.5 >3.5-4.0 >4.0-4.5 >4.5-5.0 >5.0-5.5 >1.0-1.5 >1.5-2.0 >2.0-2.5 >2.5-3.0 >3.0-3.5 >3.5-4.0 >4.0-4.5 >4.5-5.0 >5.0-5.5 Distance between Distance between Chr III probe foci (μm) Chr III probe foci (μm)

Fig. 5. Homologous pairing is independent of DSB formation. (A and B) Wild type FISH images using the telomere repeat and ChrIII probes show bouquet formation and homologous pairing in zygotene and pachytene in wild type and spo11(RNAi). DAPI panels are single z-slices; telomere and ChrIII probe panels are projections. (Scale bars, 5 μm.) (C and D) The distributions of the distances between ChrIII foci in zygotene and pachytene nuclei in wild type and spo11(RNAi) suggest that SPO11 is not required for pairing. Wild-type sycp1(RNAi) data from Fig. 3 C and D are repeated here. Pachytene/Pachytene-like nuclei had a distance <1.5 μm, n = 58) and wild-type (93.8% had a distance <1.5 μm, n = 64) animals (Fig. 5D). Together, these results suggest that homologous chromosomes pair normally in

the absence of SPO11. Moreover, knockdown of SPO11 did not sycp1+spo11(RNAi) appear to affect the localization of SYCP1 (Fig. 4 F and G), C 50 Wild type suggesting that SPO11 is not required for SC formation in pla- sycp1(RNAi) narian spermatocytes. Thus, synapsis and pairing are in- 40 sycp1+spo11(RNAi) dependent of DSB formation in S. mediterranea; this finding 30 argues strongly that, as first postulated by Gelei (5, 20, 27), it is 20 synapsis that mediates full-length homolog pairing. 10 0 DSBs Appear to Increase in Frequency by More than Twofold in sycp1 Number of RAD51 foci Zygotene Pachytene/Pachytene-like (RNAi) Planarians. TheSChasbeenfoundtobeinvolvedinthe Fig. 6. SYCP1-deficient nuclei exhibit an elevated number of RAD51 foci. control of DSB formation in other organisms. For example, in (A and B) Immunostaining of zygotene and pachytene nuclei from wild type, −/− Sycp1 mice, de Vries et al. (40) found that meiotic DSB formation sycp1(RNAi),andsycp1(RNAi); spo11(RNAi) in which RAD51 foci were ob- was normal but DSB repair was either incomplete or halted based on served. DAPI panels are single z-slices; RAD51 panels are projections. The γH2AX and ATR immunostaining. We examined whether the SC is persistence of RAD51 foci indicates that DSBs are not resolved in sycp1(RNAi). required for these functions in planarians. Based on RAD51 foci Persistent RAD51 foci likely result from programmed DSBs, as evidenced by the detection, we observed that clustered DSBs were formed in sycp1 dramatic reduction of RAD51 foci in sycp1(RNAi); spo11(RNAi).(Scalebars, (RNAi) planarians. Compared with wild-type animals, we observed 5 μm.) (C) The number of RAD51 foci in zygotene and pachytene nuclei from an elevated number of RAD51 foci in both zygotene and pachytene- wild-type, sycp1(RNAi) and sycp1(RNAi); spo11(RNAi) testes suggests that SYCP1 is required for the maturation of recombination intermediates (how- like stages (Fig. 6). At zygotene, the number of RAD51 foci per ± = ever, see Discussion). Wild-type data from Fig. 4E are repeated here, with early nucleus in sycp1(RNAi) planarians (23.3 10.3, n 129) was sig- and late zygotene combined because the knockdown of sycp1 prevents us nificantly higher than in wild-type animals (14.8 ± 6.7, n = 132) (P < from differentiating between these stages. For wild type, n = 163 nuclei in 14 0.0001) (Fig. 6 A and C). More striking, the average number of testis sections; for sycp1(RNAi), n = 161 nuclei in 16 testis sections; for sycp1 RAD51 foci in sycp1(RNAi) at pachytene-like stages was 33.2 ± 10.8 (RNAi); spo11(RNAi), n = 122 nuclei in 10 testis sections.

E5164 | www.pnas.org/cgi/doi/10.1073/pnas.1420287111 Xiang et al. Downloaded by guest on September 28, 2021 Discussion We note that the initiation of synapsis near chromosome ends PNAS PLUS Two major lessons may be drawn from this work. The first also is observed in the male meiosis of another planarian species, reflects the power of combining genomic and RNAi-knockdown Mesostoma ehrenbergii ehrenbergii (56, 57). In the three recom- analysis with immunofluorescence to elucidate the molecular bining bivalents observed in this species, both SC formation and biology of a meiotic system for which few standard forward or recombination nodule formation appear to be restricted to short reverse genetic approaches are available. In doing so we have regions corresponding to chromosomes ends. These short stretches been able to test and confirm Gelei’s model for synapsis-driven of SC are confined to a lobed region of the nucleus. Full-length homolog pairing in flatworms, at least for S. mediterranea. SC extension along the chromosome arms is not observed in this S. mediterranea thus becomes, to our knowledge, the first telo- species. Thus, it seems likely that the initiation of SC formation mere bouquet-forming organism in which SC formation has been is limited to the telomeres in at least these two planarian species, directly shown to be independent of DSB formation. However, and only in S. mediterranea is the SC allowed to extend in a there may be another example of this meiotic paradigm, namely fashion that facilitates synapsis along the full length of the Bombyx mori females, which form telomere bouquets and elab- homolog pairs. orate SCs but do not exhibit meiotic recombination (41–43). In The function of the telomere cluster in initiating SC forma- S. mediterranea, bouquet formation persists in spo11(RNAi) and tion, and thus full-length homolog pairing, in S. mediterranea is sycp1(RNAi) mutants, indicating that telomere clustering in consistent with early views of pairing initiation in which the these mutants occurs independently of DSB or SC formation, telomere cluster was thought to facilitate homolog pairing by being reminiscent of the mode of bouquet formation in yeast and bringing together the ends of homologous chromosomes in a restricted 2D space (for review, see ref. 58). However, as mammals (3, 44). We also show that meiotic DSBs preferentially a result of studies in a large number of other organisms, a dif- occur near the base of the telomere bouquet in zygotene and that ferent picture for the role of the telomere cluster has evolved for the SC is required for DSB maturation and repair. We will dis- more canonical meiosis (for review, see ref. 59). According to cuss the significance of these findings in the context of meiotic this now “classical model,” the telomere cluster and its associa- biology below. tion with the nuclear envelope function not to initiate pairing but The second lesson enumerates the minimal toolkit required rather to facilitate prophase chromosome movement. This for such a characterization: (i) a quantitative and reliable assay change in the accepted function of the telomere cluster reflects for pairing at one unique interstitial site, as well as telomeric the observation that such movements often begin too late in the repeats; (ii) a homolog for SPO11; (iii) a homolog for at least process of homolog alignment and pairing to play a major role in one SC protein; and (iv) a method for marking DSBs and fol- the pairing process and that in both budding and fission yeast lowing their maturation. such movements begin after recombination intermediates al- A critical component of this process lies in creating or possessing ready have formed. Moreover, in both fission and budding yeast, sufficiently high-quality genomic resources to allow the identifica- mutations that disrupt telomere-led movement have only modest tion of unique sequences for pairing assays as well as telomeric effects on pairing and recombination (for review, see ref. 58). sequences. Finding homologs or orthologs for the meiotic proteins Indeed, at least in the case of budding yeast, detachment of the to be used in future studies also will be critical. Identifying proteins telomeres from the nuclear envelope does not block pairing (60, residing in the central region of the SC may present a daunting 61). As pointed out by Koszul and Kleckner (59), a more rea- problem. Although TF proteins share a conserved structure con- sonable view of telomere-led motion is the generation of rapid sisting of one or more coiled-coil domains and flanking globular chromosome movements that facilitate the release of topological – domains (15, 45 47), they are difficult to identify because of a lack interlocks or other abnormal associations that improperly con- of among organisms. However, recent dem- nect bivalents by moving into and out of the telomere bouquet onstrations of characteristic microhomologies (48, 49) and insights configuration. Thus, ironically, although Gelei’s model, in which into the domain signatures of TF proteins (15, 45, 46, 50) make this telomere pairings within the base of the bouquet facilitate the goal attainable. In addition, other non-TF proteins residing in the progression of processive synapsis and full-length pairing, central region (12) also may be useful tools for such an analysis. appears to be correct in planarians (the group for which he Additionally, it would be helpful to identify meiotic genes whose proposed it), it does not appear to be broadly generalizable to products function in crossover designation and in the formation of other species—at least as the sole means for facilitating pairing axial/lateral elements and to obtain useful antibodies against them. and full-length synapsis. The long-term goal of such efforts would be to facilitate fixed and Nonetheless, the initiation of SC formation in subtelomeric live cytological assays for pairing, synapsis, and DSB fate. Al- regions at the bouquet stage and the early localization of re- though such add-ons will yield greater resolution, the basic tools combination proteins to these regions also have been seen in listed above are sufficient to provide a reasonably complete pic- barley (62, 63). However, as zygotene proceeds, numerous me- GENETICS ture of meiotic prophase in the organism of choice. However, it dial sites of SC initiation are observed also. This process may must be noted that some groups of proteins, such as those re- have parallels to the initiation of SC formation in Drosophila quired to localize DSBs (51–55), have no obvious sequence or melanogaster females, in which SC formation is observed first at structural similarities between organisms. These proteins are likely clustered centromeres (9) and appears to spread out from that to be missed by this reverse genomics approach. Finally, we note cluster supplemented by a number of sites of interstitial SC that the large fraction of the planaria body devoted to gamete formation (10). Another example of a role of subtelomeric production raises the intriguing possibility of using this system for sequences is in homolog pairing, as described by del Carmen biochemistry. For example, it is easy to imagine being able to Calderon et al. (64). In these species, SC initiation is not re- obtain sufficient material to purify structures such as SCs. stricted to the chromosome ends; rather, the ends (or the cen- tromeres in Drosophila) represent one of many sites at which A Simple Model of Full-Length Homolog Pairing in S. mediterranea. synapsis can be initiated, and the ends simply appear to act The data presented above are consistent with a model of meiotic earlier in terms of synapsis formation. prophase in planaria in which (i) telomeres are clustered on the We also have shown that DSBs primarily form near the telo- nuclear envelope to form a bouquet and (ii) the SC, and thus meres in zygotene but do so in a fashion that is independent of synapsis, is initiated at or near paired telomeric regions and SC formation. Moreover, the creation of these DSBs does not extends outward to bring the arms into full synapsis, thereby facilitate homolog pairing. We are curious about the clustering mediating the completion of chromosome pairing by pachytene. of RAD51 foci near the telomeres in early prophase. Although

Xiang et al. PNAS | Published online November 17, 2014 | E5165 Downloaded by guest on September 28, 2021 DSBs are not required for SC formation and synapsis in pla- minate the key difference of setting aside versus inducing the narians—a feature shared with C. elegans and Drosophila—pla- germ line. Understanding the molecular basis for specifying narians differ from Drosophila in that the production of wild-type cell fate and how a stem cell may choose between mitosis and numbers of DSBs does not appear to depend on the presence of meiosis to yield essentially the same output (i.e., reproduction) the SC (14). Rather, the SC appears to be critical for the repair will inform in unique ways how animals regulate cellular po- and/or maturation of DSBs. The SC also appears to be sufficient tency in vivo. for establishing and maintaining pairing in planarians even in the absence of DSBs. The observation of a more than twofold in- Meiosis, Genetics, and Evolution. All genetically tractable animal crease in the number of DSBs in pachytene nuclei that lack SC is systems available to modern biomedical research today are consistent with the recent finding in yeast that SC formation and confined to essentially two branches of life: the Deuterostomes, the stabilizing of homolog interactions acts to suppress DSB containing mice, zebrafish, Xenopus tropicalis, and humans, and formation (35, 65). We cannot exclude the possibility that the the Ecdysozoa, a superphylum of protostomes represented by observed increase in RAD51 foci reflects a failure to mature and Drosophila and C. elegans. This cohort of organisms is hardly repair DSBs in the absence of SC. representative of the vast diversity of animal forms found in nature and to which we humans are related by common evolu- Stem Cell Potency and Evolution. One of the largest collections of tionary ancestry. A branch of animal life not presently repre- reproductive strategies is found in the S. mediterranea metazoan sented by this exclusive and somewhat historically arbitrary superclade of the Lophotrochozoa (66). A large number of group of genetically tractable organisms is the Lophotrochozoa species in this group display both sexual and asexual modes of or Spiralians, a superphylum of protostome animals that is reproduction, chiefly among the many species of annelids (67, a sister group to the Ecdysozoa and the Deuterostomes. The 68), molluscs (69, 70), rotifers (71), and platyhelminthes (72–74). Lophotrochozoa comprise the largest collection of animal body However, it is surprising how little is known about the selective plans on the planet, and their remarkable biological attributes pressures that asexual reproduction may or may not have im- (e.g., prolonged longevity, regenerative properties, developmental posed on gametogenesis and on the evolution and maintenance plasticity), which likely are executed by the common genetic tools of the many sexual adaptations of this clade. Likewise, the extent possessed by all animals, remain woefully underexplored. No sig- to which sexual and asexual reproductive modes may have con- nificant advances in our understanding of life can be achieved in tributed to the evolutionary diversity of the Lophotrochozoa with the absence of genetics. Given the profound role meiosis plays in regard to genetic recombination, selfing, and their speciation is animal genetics, understanding meiosis in planarians extends the presently unknown. investigation of this fundamental process in the Lophotrochozoa Freshwater planarians possess three main modes of re- and lays the foundation for establishing genetic studies in this production: asexual, sexual, and alternating between sexual and animal and, by extension, in the entire clade. Therefore, not only asexual modes. This reproductive lifestyle ultimately depends on does our dissection of meiosis in S. mediterranea begin to address the function and fates of neoblasts, the proliferative stem cells the molecular and cellular events underpinning the evolution of found in these organisms, making the neoblast a key unit of reproductive patterns and strategies in the Metazoa; we also an- evolutionary selection. Hence, planarian biology provides a ticipate that these studies will shed light on how diverse the pro- unique opportunity to delineate in a single cell population the cess of meiosis may (or may not) be and how such diversity may autonomous and non–cell-autonomous events that lead cells be related to the evolution of genetics, form, and function in down a path of pluripotent mitotic amplification or unipotent multicellular organisms. gametogenesis production. As such, the study of meiosis in planarians offers unique opportunities to examine the evolu- Materials and Methods tionary and functional relationships among these reproduc- Planarian Care and Maintenance. Planarians were raised as described by tive strategies. Newmark and Sánchez Alvarado (75) at 20 °C. The worms were fed twice – With the exception of mammals, a central aspect of planarian a week. Once the animals reached a length of 1.5 2.0 cm, they were used biology not found in any other tractable invertebrate genetic for experiments. model system (flies and nematodes) or vertebrate model systems RNA Collection, Sequencing, and Assembly. Our transcriptome database (zebrafish, Xenopus tropicalis) is that there is no early segregation (SmedSxl_20141024, publicly available at smedgd.stowers.org) was assem- of the germ line. In all tractable genetic systems used to illumi- bled from both sexual juvenile and sexual adult planarians. RNA was nate mammalian biology, the germ line is set aside early in extracted by standard TRIzol (Ambion) methods (76). Libraries for sequenc- embryogenesis. This key developmental difference has not ing were prepared with the TruSeq RNA Sample Preparation kit v2 (RS-122- allowed us to understand better how the germ line is specified in 2001). The100-bp paired-end sequencing was completed on an Illumina mammals, a process that is likely to be essential to our un- HiSEq. 2000. Then reads were pooled and assembled using Trinity ver- derstanding of how the pluripotent state is controlled in vivo. sion trinityrnaseq-r2013-02-25 (77). The BLAST database was built using Planarians’ unique reproductive properties may help us over- BLAST version 2.2.29 (25). come these limitations. Among these properties are the follow- – ing: (i) planarian offspring are devoid of any reproductive system RNAi Plasmid Construction and RNAi Feeding. RNAi target fragments of 500 900 bp were obtained by RT-PCR. The cDNA was obtained from total RNA at birth; (ii) many species can reproduce asexually for years isolated from sexually mature animals using TRIzol Reagent (Ambion). Every before they reach sexual maturity; (iii) once they reach maturity, PCR product used for constructing RNAi plasmids was amplified with a 5′-end they are hermaphrodites; and (iv) they use the same cells (i.e., primer including an attB1 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCT-3′ se- neoblasts) to reproduce asexually and to generate the somatic quence and a 3′-end primer including an attB2 5′-GGGGACCACTTTGTACAA- tissues of the reproductive system and the germ cells in the GAAAGCTGGGT-3′ sequence for Gateway cloning. To knock down sycp1,two gonads. Taken together, these biological attributes position regions of the coding sequence were used to construct RNAi plasmids. The first planarians as a tractable model system to dissect how mitotically region was from base pairs 1–573 with two primers, 5′-ATGAAAACGTCAT- ′ ′ ′ amplifying, totipotent stem cells can be specified in a non- TTTTAAAAGG-3 and 5 -TTTGGTGAAAATTGTAATCTCCAAGGAAC-3 .The other region was from base pairs 2337–3116 with the primers 5′-TGAC- embryonic context to produce unipotent germ cells and drive GAAATGAAACTGAGAATTGAATATG-3′ and 5′-AATTTCGAAATAATTGAATCC- sexual reproduction through the meiotic production of gametes. TTTTCAGC-3′.Forspo11, full-length cDNA of the coding region was used to Although other genetically tractable model systems have allowed construct the RNAi plasmid. us to understand the similarities that exist among animals in The PCR products with attB1 and attB2 were cloned into the pPR244 plas- germline regulation, no models have been available to illu- mid (78) using Gateway Technology with Clonase II (Invitrogen). The pPR244

E5166 | www.pnas.org/cgi/doi/10.1073/pnas.1420287111 Xiang et al. Downloaded by guest on September 28, 2021 vector lacking the ccdB and ChlrR genes between attP1 and attP2 was used as ChrIII (Fig. S1), and a second probe, v31.000723, resulted in a significant PNAS PLUS a negative control. The cloned plasmids with double T7 promoters were signal that specifically labels the distal tip of one arm of ChrII (Fig. S6A).

transferred into HT115 for dsRNA production. Five milliliters of overnight- To detect the known telomere repeat, (TTAGGG)n, an oligonucleotide (5′- grown HT115 cells with target plasmid were grown in 100 mL 2× YT broth GTTAGGGTTAGGGTTAGGGTTAGG-3′) was synthesized and labeled at the 5′ (10 g/L Bacto yeast extract, 16 g/L Bacto tryptone, and 5 g/L sodium chloride) at terminus with Alexa 647 or Alexa 488 by Integrated DNA Technologies. μ μ β 37 °C with 50 g/mL kanamycin and 12.5 g/mL tetracycline. Isopropyl -D-1- For FISH of sectioned samples, slides were fixed in 4% (vol/vol) formal- thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM was added dehyde for 20 min, rinsed in 2× SSC, and then treated with 200 ng/μL RNase A – when cell density reached an OD600 value of 0.6 0.8. Four hours after IPTG was in 2× SSC for 1 h at 37 °C. After a brief rinse with 2× SSCT (SSC+0.1% Tween- − added, cells were pelleted and frozen at 80 °C for future use. 20), slides were treated sequentially in 70%, 90%, and 100% (vol/vol) eth- To prepare dsRNA food, calf liver paste was mixed with a one-third volume anol for 5 min each. After air drying, slides were kept at −20 °C overnight of planarian water, and 1 mL of the food was added to 100 mL pelleted dsRNA until use. FISH probes were applied at 4 ng/μL in hybridization buffer [2× μ bacteria mixed with 12 L of food coloring. Fifty-microliter aliquots in SSC, 50% (vol/vol) formamide, 10% (wt/vol) dextran sulfate, and 400 ng/μL − Eppendorf tubes were stored at 80 °C. Planarians (four to eight animals) sonicated salmon sperm DNA] on a glass coverslip. Probe and sample were μ were fed twice a week for 4 wk. For efficient knockdown of spo11,8 gin denatured together for 3 min on an 80 °C heat block and allowed to vitro transcribed and purified spo11 dsRNA was added to the aliquoted food reanneal overnight at 30 °C in a humid chamber. The coverslip was removed, just before feeding. and slides were washed twice with 50% (vol/vol) formamide/2× SSCT for 10 min at 30 °C and then twice in 2× SSCT at room temperature. Slides then Cryosectioning of Planarian Samples. Planarian parasagittal sections were were stained with 500 ng/mL DAPI for 10 min. After two washings in 2× produced for DAPI staining, immunostaining, and FISH. Animals first were SSCT, slides were mounted in Vectashield. immersed in Tissue-Tek OCT medium (Ted Pella, Inc.) in a Tissue-Tek mold. For metaphase spreads, animals were cut into 1- or 2-mm segments and were After the worm stopped moving, the mold was kept at −60 °C until frozen. allowed to regenerate under normal growth conditions [1× Montjuic buffer The frozen worm was kept −20 °C while being sliced into 10-μm sections. The (1.6 mM NaCl, 1.0 mM CaCl , 1.0 mM MgSO , 0.1 mM MgCl ,0.1mMKCl, sections on slides were kept at −20 °C for future use. 2 4 2 1.2 mM NaHCO3, pH 7.0), 20 °C] for 3 d. The fragments were soaked in 5 mM colchicine/1× Montjuic solution for 8–12 h overnight, transferred to 1% so- Antibody Preparations. For antibody preparation, recombinant proteins dium citrate at 37 °C for 20 min, and fixed in 3:1 methanol:glacial acetic acid at SMED-RAD51 (full-length) and SMED-SYCP1 (amino acids 1–300) were −20 °C for at least 2 h (and for up to several months). The fragments were expressed and purified. The cDNAs of the Smed-rad51 and Smed-sycp1 placed in 60% (vol/vol) glacial acetic acid until degraded and homogenized by genes were obtained by RT-PCR. The cloned genes were expressed in pipetting. The solution was dropped onto clean slides and allowed to air dry; Escherichia coli, and the purified proteins were used to develop antibodies. slides were stored at −20 °C overnight. Slides were treated sequentially in Antibodies were generated by Cocalico Biologicals and affinity purified. 70%, 90%, and 100% (vol/vol) ethanol and kept as above until use. FISH was Fluorescent secondary antibodies conjugated with Alexa 488, 555, and 647 conducted as described above. were purchased from Invitrogen or Jackson ImmunoResearch.

Identification of Smed-spo11. A BLAST search of SmedGD identified a 93-bp Immunostaining and Microscopy. Cryosectioned samples were fixed in 4% (vol/ DNA fragment encoding 31 amino acids with homology to mouse SPO11. To vol) formaldehyde for 20 min at room temperature. Fixed sections were find the 3′ end of Smed-spo11,3′ RACE was performed using a primer based washed twice in PBS+0.5% Triton X-100 (PBS-TX0.5) for 10 min each. The on the 93-bp DNA sequence identified. The 5′ end of Smed-spo11 was fixed sections then were subjected to antigen retrieval with Trilogy reagent obtained by PCR using the primer GACGGTCTTATCGAAATCTATATAAATCTTAT, (Cell Marque) according to the manufacturer’s protocol. Slides were in- the planarian spliced-leader 1 (Smed SL-1) sequence (79), and the primer cubated in PBS-TX0.5 for 30 min and transferred to a blocking solution (0.5% AGAACCATATTTGAACGAACAGTAAATTG from the known sequence. IgG-free BSA and PBS-TX0.5) for 1.5 h. Primary antibodies were diluted in blocking solution and applied for 1 h (or overnight at 4 °C). Slides were washed with alternating changes of PBS-TX0.5 and PBS+0.1% Tween-20 Protein Alignments. Protein alignments for SPO11 and SYCP1 were done in (PBS-TW). Secondary antibodies were diluted in blocking solution and ap- JalView (80) using ClustalX with default settings (81) for SYCP1 and Clustal plied for 1 h, and slides were washed again with PBS-TX0.5 and PBS-TW. Omega (82) for SPO11. Sequences were obtained from the NCBI Protein DAPI (500 ng/mL in PBS-TW) was applied for 10 min. After two 10-min database (www.ncbi.nlm.nih.gov/protein/). The conservation threshold for washes with PBS-TW, slides were mounted in Vectashield mounting medium. A colored shading of the residues was set to 50%, and sequence similarity was DeltaVision microscopy system (Applied Precision) was used to capture images, determined by eight groups: identical, aliphatic/hydrophobic, aromatic, which then were deconvolved with softWoRx v.25 software and projected as positive, negative, hydrophilic, proline/glycine, and cysteine. multiple z-stacks. X-Ray Irradiation. Planarians were exposed to X-ray radiation at 1.0 Gy/min for DNA Probe Labeling and FISH. To locate a suitable region for a euchromatic a dosage of 10 Gy. After irradiation, the worms were kept at 20 °C for 5 h. DNA probe, we used the S. mediterranea genome database (smedgd.stowers. Then the irradiated worms were frozen, sectioned, and immunostained as org) to identify contigs covering regions of about 100 kb that contained described above. few large repetitive repeats and that coded for several genes. We tested four contigs that fit these criteria: v31.000191 from base pairs 106,000– ACKNOWLEDGMENTS. We thank Harry Scherthan, Peter Baumann, Sue

210,000; v31.000323 from base pairs 19,000–114,800; v31.000325 from base Jaspersen, and Cathy Lake for critical reading of the manuscript; members of GENETICS pairs 30,150–130,400; and v31.000723 from base pairs 40,000–143,000. For the R.S.H. laboratory for helpful comments on the manuscript; Angela Miller for editorial and graphics assistance; Michael Anderson for technical support; each region, we designed 50 pairs of primers to amplify 50 ∼2-kb fragments. and the Stowers Institute for Medical Research Aquatics and Histology facili- PCR products were gel purified and pooled together in equal amounts. ties. A.S.A. and R.S.H. are supported by the Stowers Institute for Medical Re- Fluorophore labeling was performed as described in the ULYSIS Nucleic Acid search. A.S.A. also is supported by the Howard Hughes Medical Institute. R.S.H. Labeling Kit (Invitrogen) instructions. One probe, v31.000191, produced is an American Cancer Society Research Professor supported by American a significant FISH signal that specifically marks the middle of the arm of Cancer Society Award RP-05-086-06DDC.

1. de Massy B (2013) Initiation of meiotic recombination: How and where? Conservation 7. McKim KS, et al. (1998) Meiotic synapsis in the absence of recombination. Science and specificities among eukaryotes. Annu Rev Genet 47:563–599. 279(5352):876–878. 2. Kleckner N (2006) Chiasma formation: Chromatin/axis interplay and the role(s) of the 8. Carpenter AT (1975) Electron microscopy of meiosis in Drosophila melanogaster fe- synaptonemal complex. Chromosoma 115(3):175–194. males. I. Structure, arrangement, and temporal change of the synaptonemal complex 3. Trelles-Sticken E, Loidl J, Scherthan H (1999) Bouquet formation in budding yeast: in wild-type. Chromosoma 51(2):157–182. Initiation of recombination is not required for meiotic telomere clustering. J Cell Sci 9. Takeo S, Lake CM, Morais-de-Sá E, Sunkel CE, Hawley RS (2011) Synaptonemal com- 112(Pt 5):651–658. plex-dependent centromeric clustering and the initiation of synapsis in Drosophila 4. Rasmussen SW, Holm PB (1980) Mechanics of meiosis. Hereditas 93(2):187–216. oocytes. Curr Biol 21(21):1845–1851. 5. Scherthan H (2001) A bouquet makes ends meet. Nat Rev Mol Cell Biol 2(8):621–627. 10. Tanneti NS, Landy K, Joyce EF, McKim KS (2011) A pathway for synapsis initiation 6. Lee CY, Conrad MN, Dresser ME (2012) Meiotic chromosome pairing is promoted by during zygotene in Drosophila oocytes. Curr Biol 21(21):1852–1857. telomere-led chromosome movements independent of bouquet formation. PLoS 11. Christophorou N, Rubin T, Huynh JR (2013) Synaptonemal complex components Genet 8(5):e1002730. promote centromere pairing in pre-meiotic germ cells. PLoS Genet 9(12):e1004012.

Xiang et al. PNAS | Published online November 17, 2014 | E5167 Downloaded by guest on September 28, 2021 12. Page SL, et al. (2008) Corona is required for higher-order assembly of transverse fil- 48. Fraune J, Brochier-Armanet C, Alsheimer M, Benavente R (2013) Phylogenies of aments into full-length synaptonemal complex in Drosophila oocytes. PLoS Genet central element proteins reveal the dynamic evolutionary history of the mammalian 4(9):e1000194. synaptonemal complex: Ancient and recent components. Genetics 195(3):781–793. 13. Page SL, Hawley RS (2001) c(3)G encodes a Drosophila synaptonemal complex protein. 49. Fraune J, Wiesner M, Benavente R (2014) The synaptonemal complex of basal Genes Dev 15(23):3130–3143. metazoan hydra: More similarities to vertebrate than invertebrate meiosis model 14. Mehrotra S, McKim KS (2006) Temporal analysis of meiotic DNA double-strand break organisms. J Genet Genomics 41(3):107–115. formation and repair in Drosophila females. PLoS Genet 2(11):e200. 50. Schild-Prüfert K, et al. (2011) Organization of the synaptonemal complex during 15. Collins KA, et al. (2014) Corolla is a novel protein that contributes to the architecture meiosis in Caenorhabditis elegans. Genetics 189(2):411–421. of the synaptonemal complex of Drosophila. Genetics 198(1):219–228. 51. Kumar R, Bourbon HM, de Massy B (2010) Functional conservation of Mei4 for meiotic 16. Dernburg AF, et al. (1998) Meiotic recombination in C. elegans initiates by a con- DNA double-strand break formation from yeasts to mice. Genes Dev 24(12): served mechanism and is dispensable for homologous chromosome synapsis. Cell 1266–1280. 94(3):387–398. 52. Lake CM, Nielsen RJ, Hawley RS (2011) The Drosophila zinc finger protein trade 17. MacQueen AJ, et al. (2005) Chromosome sites play dual roles to establish homologous embargo is required for double strand break formation in meiosis. PLoS Genet 7(2): synapsis during meiosis in C. elegans. Cell 123(6):1037–1050. e1002005. 18. Phillips CM, Dernburg AF (2006) A family of zinc-finger proteins is required for 53. Liu H, Jang JK, Kato N, McKim KS (2002) mei-P22 encodes a chromosome-associated chromosome-specific pairing and synapsis during meiosis in C. elegans. Dev Cell 11(6): protein required for the initiation of meiotic recombination in Drosophila mela- – 817–829. nogaster. Genetics 162(1):245 258. 19. White MJD (1977) Animal Cytology and Evolution (Cambridge Univ Press, London), 54. Rosu S, et al. (2013) The C. elegans DSB-2 protein reveals a regulatory network that 3rd Ed. controls competence for meiotic DSB formation and promotes crossover assurance. 20. Gelei J (1921) Weitere Studien über die Oogenese des Dendrocoelum lacteum. II. Die PLoS Genet 9(8):e1003674. Längskonjugation der Chromosomen. Arch Zellforsch 16:88–169. 55. Stamper EL, et al. (2013) Identification of DSB-1, a protein required for initiation of 21. Newmark PA, Wang Y, Chong T (2008) Germ cell specification and regeneration in meiotic recombination in Caenorhabditis elegans, illuminates a crossover assurance planarians. Cold Spring Harb Symp Quant Biol 73:573–581. checkpoint. PLoS Genet 9(8):e1003679. 22. Chong T, Stary JM, Wang Y, Newmark PA (2011) Molecular markers to characterize 56. Oakley HA, Jones GH (1982) Meiosis in Mesostoma ehrenbergii ehrenbergii (Turbel- the hermaphroditic reproductive system of the planarian Schmidtea mediterranea. laria, Rhabdocoela). I. Chromosome pairing, synaptonemal complexes and chiasma – BMC Dev Biol 11:69. localization in spermatogenesis. Chromosoma 85(3):311 322. 23. Chretien JH (2011) Characterization of Spermatogenesis in the Planarian S. medi- 57. Croft JA, Jones GH (1989) Meiosis in Mesostoma ehrenbergii ehrenbergii. IV. Re- terranea. PhD dissertation. (University of California, Berkeley). combination nodules in spermatocytes and a test of the correspondence of late re- – 24. Schmekel K, et al. (1996) Organization of SCP1 protein molecules within synaptone- combination nodules and chiasmata. Genetics 121(2):255 262. mal complexes of the rat. Exp Cell Res 226(1):20–30. 58. Zickler D, Kleckner N (1998) The leptotene-zygotene transition of meiosis. Annu Rev – 25. Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: A new generation of protein Genet 32:619 697. 59. Koszul R, Kleckner N (2009) Dynamic chromosome movements during meiosis: A way database search programs. Nucleic Acids Res 25(17):3389–3402. – 26. Fraune J, et al. (2012) Hydra meiosis reveals unexpected conservation of structural to eliminate unwanted connections? Trends Cell Biol 19(12):716 724. 60. Lui DY, Cahoon CK, Burgess SM (2013) Multiple opposing constraints govern chro- synaptonemal complex proteins across metazoans. Proc Natl Acad Sci USA 109(41): mosome interactions during meiosis. PLoS Genet 9(1):e1003197. 16588–16593. 61. Trelles-Sticken E, Dresser ME, Scherthan H (2000) Meiotic telomere protein Ndj1p is 27. Gelei J (1922) Weitere Studien über die Oogenese des Dendrocoelum lacteum. III. Die required for meiosis-specific telomere distribution, bouquet formation and efficient Konjugations-frage der Chromosomen in der Literature und meine Befunde. Arch homologue pairing. J Cell Biol 151(1):95–106. Zellforsch 16:299–370. 62. Higgins JD, Osman K, Jones GH, Franklin FC (2014) Factors Underlying Restricted 28. Tan TC, et al. (2012) Telomere maintenance and telomerase activity are differentially Crossover Localization in Barley Meiosis. Annu Rev Genet 48:29–47. regulated in asexual and sexual worms. Proc Natl Acad Sci USA 109(11):4209–4214. 63. Higgins JD, et al. (2012) Spatiotemporal asymmetry of the meiotic program underlies 29. Beliveau BJ, et al. (2012) Versatile design and synthesis platform for visualizing ge- the predominantly distal distribution of meiotic crossovers in barley. Plant Cell 24(10): nomes with Oligopaint FISH probes. Proc Natl Acad Sci USA 109(52):21301–21306. 4096–4109. 30. Loidl J, Klein F, Scherthan H (1994) Homologous pairing is reduced but not abolished 64. del Carmen Calderon M, Rey M-D, Cabrera A, Prieto P (2014) The subtelomeric region in asynaptic mutants of yeast. J Cell Biol 125(6):1191–1200. is important for chromosome recognition and pairing during meiosis. Sci Rep 4:6488. 31. Weiner BM, Kleckner N (1994) Chromosome pairing via multiple interstitial inter- 65. Thacker D, Mohibullah N, Zhu X, Keeney S (2014) Homologue engagement controls actions before and during meiosis in yeast. Cell 77(7):977–991. meiotic DNA break number and distribution. Nature 510(7504):241–246. 32. Peoples TL, Dean E, Gonzalez O, Lambourne L, Burgess SM (2002) Close, stable ho- 66. Extavour CG (2007) Gray anatomy: Phylogenetic patterns of somatic gonad structures molog juxtaposition during meiosis in budding yeast is dependent on meiotic re- and reproductive strategies across the Bilateria. Integr Comp Biol 47(3):420–426. combination, occurs independently of synapsis, and is distinct from DSB-independent 67. Rasmussen E (1953) Asexual reproduction in Pygospio elegans claparède (Polychaeta pairing contacts. Genes Dev 16(13):1682–1695. sedentaria). Nature 171(4365):1161–1162. 33. Boateng KA, Bellani MA, Gregoretti IV, Pratto F, Camerini-Otero RD (2013) Homol- 68. Martin EA (1933) Polymorphism and methods of asexual reproduction in the annelid ogous pairing preceding SPO11-mediated double-strand breaks in mice. Dev Cell Dodecaceria, of vineyard sound. Biol Bull 65(1):99–105. – 24(2):196 205. 69. Thiriot-Quiévreux C, Soyer F, Bovée F, Albert P (1988) Unusual chromosome com- 34. Ishiguro K, et al. (2014) Meiosis-specific cohesin mediates homolog recognition in plement in the brooding bivalve Lasaea consanguinea. Genetica 76(2):143–151. – mouse spermatocytes. Genes Dev 28(6):594 607. 70. Neiman M, Larkin K, Thompson AR, Wilton P (2012) Male offspring production by – 35. Zhu X, Keeney S (2014) Zip it up to shut it down. Cell Cycle 13(14):2157 2158. asexual Potamopyrgus antipodarum, a New Zealand snail. Heredity (Edinb) 109(1): 36. Cantarel BL, et al. (2008) MAKER: An easy-to-use annotation pipeline designed for 57–62. – emerging model organism genomes. Genome Res 18(1):188 196. 71. Becks L, Agrawal AF (2010) Higher rates of sex evolve in spatially heterogeneous 37. Rossi A, Ross EJ, Jack A, Sánchez Alvarado A (2014) Molecular cloning and charac- environments. Nature 468(7320):89–92. terization of SL3: A stem cell-specific SL RNA from the planarian Schmidtea medi- 72. Sonneborn TM (1930) Genetic studies on Stenostomum incaudatum (nov spec.). J Exp – terranea. Gene 533(1):156 167. Zool 57(1):57–108. 38. Malik SB, Ramesh MA, Hulstrand AM, Logsdon JM, Jr (2007) Protist homologs of the 73. Kenk R (1937) Sexual and asexual reproduction in Euplanaria tigrina (Girard). Biol Bull meiotic Spo11 gene and topoisomerase VI reveal an evolutionary history of gene 73(2):280–294. – duplication and lineage-specific loss. Mol Biol Evol 24(12):2827 2841. 74. Kobayashi K, Hoshi M (2002) Switching from asexual to sexual reproduction in the 39. Inagaki A, Schoenmakers S, Baarends WM (2010) DNA double strand break repair, planarian Dugesia ryukyuensis: Change of the fissiparous capacity along with the chromosome synapsis and transcriptional silencing in meiosis. Epigenetics 5(4): sexualizing process. Zoolog Sci 19(6):661–666. 255–266. 75. Newmark PA, Sánchez Alvarado A (2000) Bromodeoxyuridine specifically labels the 40. de Vries FA, et al. (2005) Mouse Sycp1 functions in synaptonemal complex assembly, regenerative stem cells of planarians. Dev Biol 220(2):142–153. meiotic recombination, and XY body formation. Genes Dev 19(11):1376–1389. 76. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guani- 41. Sturtevant AH (1915) No Crossing Over in the Female of the Silkworm Moth. Am Nat dinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162(1):156–159. 49(577):42–44. 77. Haas BJ, et al. (2013) De novo transcript sequence reconstruction from RNA-seq using 42. Rasmussen SW (1976) The meotic prophase in Bombyx mori females analyzed by the Trinity platform for reference generation and analysis. Nat Protoc 8(8):1494–1512. three dimensional reconstructions of synaptonemal complexes. Chromosoma 54(3): 78. Reddien PW, Bermange AL, Murfitt KJ, Jennings JR, Sánchez Alvarado A (2005) 245–293. Identification of genes needed for regeneration, stem cell function, and tissue 43. Rasmussen S, Holm P (1979) Chromosome pairing in autotetraploid Bombyx females. homeostasis by systematic gene perturbation in planaria. Dev Cell 8(5):635–649. Mechanism for exclusive bivalent formation. Carlsberg Res Commun 44(2):101–125. 79. Zayas RM, Bold TD, Newmark PA (2005) Spliced-leader trans-splicing in freshwater 44. Liebe B, et al. (2006) Mutations that affect meiosis in male mice influence the dy- planarians. Mol Biol Evol 22(10):2048–2054. namics of the mid-preleptotene and bouquet stages. Exp Cell Res 312(19):3768–3781. 80. Waterhouse AM, Procter JB, Martin DM, Clamp M, Barton GJ (2009) Jalview Version 45. Zickler D, Kleckner N (1999) Meiotic chromosomes: Integrating structure and func- 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics tion. Annu Rev Genet 33:603–754. 25(9):1189–1191. 46. Page SL, Hawley RS (2004) The genetics and molecular biology of the synaptonemal 81. Larkin MA, et al. (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23(21): complex. Annu Rev Cell Dev Biol 20:525–558. 2947–2948. 47. de Boer E, Heyting C (2006) The diverse roles of transverse filaments of synaptonemal 82. Sievers F, et al. (2011) Fast, scalable generation of high-quality protein multiple complexes in meiosis. Chromosoma 115(3):220–234. sequence alignments using Clustal Omega. Mol Syst Biol 7:539.

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