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ScienceDirect Journal of Genetics and Genomics 41 (2014) 107e115

JGG REVIEW

The Synaptonemal Complex of Basal Metazoan Hydra: More Similarities to Vertebrate than Invertebrate Meiosis Model Organisms Johanna Fraune*, Miriam Wiesner, Ricardo Benavente* Department of Cell and Developmental Biology, Biocenter, University of Wu¨rzburg, D-97074 Wu¨rzburg, Germany Received 30 September 2013; revised 18 December 2013; accepted 20 January 2014 Available online 20 February 2014

ABSTRACT The synaptonemal complex (SC) is an evolutionarily well-conserved structure that mediates chromosome synapsis during prophase of the first meiotic division. Although its structure is conserved, the characterized protein components in the current metazoan meiosis model systems (Drosophila melanogaster, Caenorhabditis elegans, and Mus musculus) show no sequence homology, challenging the question of a single evolutionary origin of the SC. However, our recent studies revealed the monophyletic origin of the mammalian SC protein components. Many of them being ancient in Metazoa and already present in the cnidarian Hydra. Remarkably, a comparison between different model systems disclosed a great similarity between the SC components of Hydra and mammals while the proteins of the ecdysozoan systems (D. melanogaster and C. elegans) differ significantly. In this review, we introduce the basal-branching metazoan species Hydra as a potential novel invertebrate model system for meiosis research and particularly for the investigation of SC evolution, function and assembly. Also, available methods for SC research in Hydra are summarized. KEYWORDS: Meiosis; Synaptonemal complex; Hydra; Evolution

INTRODUCTION Meiosis is a cell division type that results in the production of haploid germ cells. The two successive meiotic divisions are evolutionarily highly conserved and nearly ubiquitous in sexually reproducing organisms. One of the key structures of meiosis is the synaptonemal complex (SC) that assembles during prophase I. During this phase, it mediates the stable pairing (synapsis) of homologous chromosomes, which is necessary for the accurate reduction of the chromosome set to a haploid state (haploidization). Failures during this process can lead to aneuploid germ cells or even induce apoptosis (Hassold and Hunt, 2001; Handel and Schimenti, 2010). The structure of the SC is highly conserved in evolution and consists of three domains (Heyting, 1996; Page and Hawley, * Corresponding authors. Tel: þ49 931 3184254, fax: þ49 931 3184252. E-mail addresses: [email protected] (J. Fraune); [email protected] (R. Benavente).

2004): (1) the lateral elements (LEs) which form early during leptotene and become clearly visible in zygotene along each chromosome; (2) the transverse filaments (TFs) which consist of large coiled-coil proteins that assemble between the two LEs of each homologous chromosome pair connecting them stably along their entire length similar to a zipper. This synapsis is initiated during zygotene and completed in pachytene when all chromosomes are stably paired. In diplotene the chromosomes desynapse again by the disassembly of the TFs. (3) The central element (CE) which runs parallel to the LEs in the center of the SC, most likely supporting the interactions of opposing TFs (Schmekel and Daneholt, 1995; Heyting, 1996; Page and Hawley, 2004). Despite of the evolutionary conservation of the SC structure, the characterized SC protein components of current metazoan meiosis model organisms (i.e., Mus musculus, Drosophila melanogaster and Caenorhabditis elegans) lack detectable evolutionary relationship (Page and Hawley, 2004; Bogdanov et al., 2007). Both numbers and sequences of SC

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protein components vary between the SCs of these model organisms. This apparent discrepancy between structural conservation of the SC and the divergent protein composition in the major metazoan meiosis model organisms has been a matter of recent investigations (Fraune et al., 2012a, 2013). Here, we will summarize the recent data that have provided new insight on the evolutionary history of SC proteins in metazoans. As many of the relevant data have been obtained in the basal metazoan Hydra, we will also discuss whether this species may become a suitable model organism for metazoan SC research.

THE EVOLUTION OF THE METAZOAN SC In mammals, seven proteins are described to be part of the SC. SYCP2 and SYCP3 are the constituents of the LEs (Lammers et al., 1994; Offenberg et al., 1998). The large coiled-coil protein SYCP1 is the only known component of the TFs (Meuwissen et al., 1992) whereas the four small proteins SYCE1, SYCE2, SYCE3 and Tex12 are specific to the CE (Costa et al., 2005; Hamer et al., 2006; Schramm et al., 2011). In D. melanogaster, the proteins ORD and C(2)M form the LEs, C(3)G is the protein component of the TFs and CONA is likely a CE protein (Bickel et al., 1996; Page and Hawley, 2001; Manheim and McKim, 2003; Webber et al., 2004; Page et al., 2008; Lake and Hawley, 2012). In contrast, HIM-3 is the LE protein of C. elegans and SYP-1, SYP-2, SYP-3 and SYP-4 were shown to be localized to the central region (CR) of the SC that encompasses the TFs and the CE (MacQueen et al., 2002; Colaiacovo et al., 2003; Smolikov et al., 2007, 2009; Hawley, 2011; Schild-Pru¨fert et al., 2011). This obvious discrepancy between the structural conservation and the protein diversity of the SC prompted us to investigate the phylogeny of the mammalian SC components more carefully. Our work revealed unexpected monophyletic origins of SYCP1, SYCP3, SYCE1, SYCE2, SYCE3 and Tex12 which, however, occurred at different time points during the evolution of metazoan (Fraune et al., 2012a, 2013). The TF protein SYCP1, the LE protein SYCP3 and the CE proteins SYCE2 and Tex12 already exist in the cnidarian species Hydra at the very base of metazoan life whereas the CE proteins SYCE1 and SYCE3 seem to have evolved later in the last common ancestor of Bilateria and vertebrates, respectively (Fraune et al., 2012a, 2013). The reason for the addition of further CE components during metazoan evolution is still unknown. However, the more ancient CE proteins SYCE2 and Tex12, which tightly interact with each other to form the elongation complex in mammals (Davies et al., 2012; Fraune et al., 2012b), might be indispensable for the functionality of the basal SC due to the interplay between SC assembly and homologous recombination, which occurs in many species (Padmore et al., 1991; Moens et al., 2007). In mammals, only the conserved components SYCP1, SYCP3 and SYCE2 have been known to be able to interact with RAD51 and DMC1, two markers for early recombination nodules (Tarsounas et al., 1999; Moens et al., 2002; Bolcun-Filas et al., 2009).

Our bioinformatics and expression studies support the model that the SC arose only once in Metazoa but has undergone a dynamic evolutionary history during which new components were recruited into the structure. Additionally, diversification of SC proteins may have occurred. This is most likely the case in the clade of Ecdysozoa: our phylogenetic analysis could not identify any ecdysozoan sequences related to mammalian SC proteins with the exception of few distantly related crustacean sequences of SYCP1 and SYCE2. Furthermore, no protein sequences could be detected outside the genus of Drosophila and Caenorhabditis, which were homologous to their characteristic SC protein components (Fraune et al., 2012a, 2013). Available evidence suggests that the protein components of the D. melanogaster and C. elegans SCs must either be of more recent origin or, more likely, have highly diverged specifically in these genera (Fraune et al., 2012a, 2013). CAN HYDRA BECOME A FULLY FLEDGED MODEL ORGANISM FOR RESEARCH ON THE SC? The competence of Hydra to reproduce sexually and the presence of the ancient SC allowed us to consider the polyp as a potential invertebrate system to study meiotic questions, combining the favorable simplicity of an invertebrate model with the ability to draw general conclusions about the SC and its evolution. What is known about Hydra gametogenesis and meiosis? What methods are available for SC research in Hydra? These questions will be recapitulated in this section. Hydra e a versatile model system since the 18th century Researchers have used Hydra as a model organism for more than 250 years, studying classical biological questions such as tissue regeneration, embryogenesis, neurogenesis, body axis patterning, cell signaling and symbiosis as well as ageing and multipotency of stem cells. Furthermore, the fast development of new genomic and proteomic methods has turned Hydra into a favored model system for molecular biologists and geneticists since the late 20th century. Current research topics include the maintenance of tissue homeostasis, innate immunity and stress response as well as molecular mechanisms of senescence and stemness (Galliot, 2012). Finally, the publication of the first sequenced Hydra genome (Chapman et al., 2010) further increased the number of molecular questions that were answered by using this little fresh water polyp. In particular, issues of metazoan evolution could be approached. Evolutionary analyses revealed the conservation of a large number of gene families between cnidarian and vertebrates that were lost or highly diverged in ecdysozoan species, e.g., arthropods and nematodes (Galliot, 2012). Hydra is at the base of metazoan life. It belongs to the old phylum of Cnidaria, which is a sister group of Bilateria (Fig. 1). It attracts scientists with its simple body plan consisting of only two epithelia layers (ectoderm and endoderm) that encase the gastric cavity. A tentacle ring around the mouth opening at the top is used for active capturing of its prey. With an adhesive foot, Hydra can stick to the solid ground of fresh

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Fig. 1. Diagram of metazoan evolution. The diagram illustrates the ancient character of Hydra (Cnidaria) and its evolutionary relationship to the mouse (Tetrapoda) as well as to the other model organisms Drosophila melanogaster (Hexapoda) and Caenorhabditis elegans (Nematoda).

water (Steele, 2012). Under laboratory conditions, Hydra can easily be cultured in appropriate medium at temperatures between 18 C and room temperature. Adjusted to the need, the polyps are fed a few times a week with Artemia nauplii, followed by a short cleaning step and the exchange of the medium. Indeed, no cell culture system is available for Hydra cells, leaving only the in vivo manipulation of animals for experiment. Nonetheless, culturing Hydra in large number is possible and more detailed instructions are provided by Lenhoff and Brown (1970). Hydra has three different stem cell lineages. While two stem cell lineages give rise to the endoderm (endodermal stem cell lineage) and the ectoderm (ectodermal stem cell lineage), the third, called interstitial stem cell lineage, differentiates to nematocytes, nerve cells, gland cells and gametes (Bosch et al., 2010). Even though Hydra usually reproduces asexually by budding, extrinsic and/or intrinsic signals can lead to the formation of gonads and a switch to the sexual reproduction mode (Littlefield et al., 1991; Kuznetsov et al. 2001). However, little is known about gametogenesis and sex determination in Hydra. Depending on the Hydra strain used, egg and sperm differentiation can be induced in the laboratory by low temperature or starvation (Kuznetsov et al. 2001; Wittlieb

et al. 2006). The initial signals which lead to the formation of gonads and the germ line are unknown. From former analysis on Hydra oligactis and H. magnipapillata, it was speculated that a self-renewing subpopulation of interstitial cells exists which is restricted to the developmental fate of sperms, while another is committed to the formation of eggs and the somatic cell types (Littlefield, 1985; NishimiyaFujisawa and Sugiyama, 1993). The sexual phenotype of the animal is dependent on the sex of the interstitial cell, but independent on the genetic sex of the epithelial cells. Some Hydra species even show the phenomenon of sex reversal (Bosch and David, 1986). However, the existence of a heteromorphic sex chromosome pair could not be detected in the karyotype of H. magnipapillata chromosomes. Instead, this revealed a karyotype with 30 two-armed meta- or submetacentric chromosomes (Anokhin et al., 2010). In spermatogenesis, interstitial cells that are restricted to sperm differentiation locally accumulate in the intraepithelial space, lift the ectoderm and form the conical swellings of the testes along the body column. The segmentation of the testes into distinct chambers by epithelial protrusions is observed in longitudinal optical sections through a fluorescently labeled Hydra testis (Fig. 2). In these compartments, the interstitial

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cells synchronously undergo the differentiation pathway from spermatogonia via spermatocytes and spermatids to mature spermatozoa, which are released into the water (Kuznetsov et al., 2001). Because epithelial cells and germ cells are in close contact, it was suggested that the somatic tissue plays an active role in controlling the microenvironment of the differentiating sperm precursors, as do Sertoli cells in the mammalian testis (Kuznetsov et al., 2001). During oogenesis, interstitial cells that are committed to egg differentiation accumulate beneath the ectoderm. One of these cells, which are located in the center of the cell aggregate, develops into an oocyte, while the other cells become nurse cells. The nurse cells finally undergo apoptosis and are phagocytosed by the oocyte (Miller et al., 2000). Because only few oocytes are present at a time, male gametogenesis is more suitable for meiosis research than oogenesis. Inventory of Hydra SC protein components One of our main concerns in meiosis research has been the composition, assembly and function of the mammalian SC. Many general questions were answered by studying mouse models and the phenotypes of several SC knock-out lines (Yuan et al., 2000; de Vries et al., 2005; Yang et al., 2006; Bolcun-Filas et al., 2007, 2009; Hamer et al., 2008; Schramm et al., 2011). However, evolutionary aspects were neglected because of the missing discernible relationship to

the invertebrate systems of D. melanogaster and C. elegans. Our recent work on Hydra revealed that SC proteins were monophyletic from Cnidaria to mammals (Fraune et al., 2012a, 2013), providing the opportunity to study orthologs of the mammalian SC proteins in the simple context of the Hydra organism. Four SC proteins have been characterized in H. vulgaris (strain AEP): HySYCP1, HySYCP3, HySYCE2, HyTex12 (Fraune et al., 2012a, 2013) (Fig. 3). As reported for other model organisms, these proteins are specifically expressed during meiosis. HySYCP3 is 237 aa long and has a small coiled-coil domain at the C-terminal end. Like its mammalian ortholog, it localizes to the LEs of the SC and is detectable along the chromosome axes from leptotene to diakinesis. HySYCP1 consists of 1016 aa and has extended coiled-coil domains in the central region of the protein. The N- and Ctermini are non-helical. It specifically localizes to the synapsed regions of the chromosomes from zygotene until diplotene and therefore is a specific marker for the central region (Fraune et al., 2012a). Because HySYCP1 resembles other TF proteins in sequence, size and predicted secondary structure (Meuwissen et al., 1992; Page and Hawley, 2001), it might be a cnidarian ortholog to the TF protein of other SCs. HySYCE2 and HyTex12 are small proteins of 152 aa and 111 aa, respectively. Both proteins contain short coiled-coil motifs at their Cterminal ends. Like HySYCP1, the proteins exclusively localize to regions between synapsed LEs, but in a more punctate pattern in contrast to the continuous localization observed for HySYCP1 (Fraune et al. 2012a, 2013). This was likewise already observed for the CE-specific mammalian orthologs SYCE2 and Tex12 (Costa et al., 2005; Hamer et al., 2006). In summary, our recent studies demonstrated that Hydra possesses proteins belonging to all three conserved domains of the SC, namely LEs (HySYCP3), the TFs (HySYCP1) and the CE (HySYCE2 and HyTex12) (Fig. 4) (Fraune et al., 2012a, 2013). The existence of other unidentified components cannot be excluded a priori. Available methods for Hydra SC research As mentioned above, our bioinformatic and expression studies have provided compelling evidence for the

Fig. 2. Whole mount fluorescence microscopy of a Hydra testis. Different developmental stages of sperm precursor cells can be distinguished by the size of their stained nuclei (blue). Spermatogonia and spermatocytes can be found in the basal layers of the testis while spermatids and spermatozoa localize closer to the tip. The sperm differentiation occurs in a spatial order from the base to the tip of the testis. The testis itself is separated into distinct chambers by epithelial protrusions indicated by the phalloidin-staining of the cellular actin skeleton (red). Scale bar, 50 mm.

Fig. 3. Schematic representation of the inventory of Hydra vulgaris (strain AEP) SC components. The length of the proteins (given in number of amino acids) as well as the extension and position of coiled-coil domains according to the Lupas algorithm (green boxes) are provided.

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Fig. 4. Schematic illustration of the Hydra SC and the localization of its components according to available immunolocalization data. Similar schemes of fly, worm and mouse SCs are indicated in Hawley (2011) and Fraune et al. (2012b).

monophyletic origin of the metazoan SC (Fraune et al., 2012a, 2013). Here, we will summarize available methodological approaches to comparative studies between mammalian and cnidarian SCs. Meiosis-specific expression of potential SC proteins in mouse is usually proven by RT-PCR and Western blot analysis on different tissue fractions such as skin, liver, kidney, heart, muscle, brain and gonads (Schramm et al., 2011). Despite of its lack of differentiated organs, Hydra can be separated into different body fractions, namely head, mid-piece, foot and gonads. In particular in male animals, it is easy to separate the numerous conical swellings that represent the testes from the body column. Whole mount in situ hybridization, which is simply done in Hydra, complements the available methods for the analysis of the testis-specific expression of the SC candidate proteins (Fraune et al., 2012a, 2013). A further standard tool for studying meiotic proteins is the microscopic analysis at both the electron microscopic as well as at the light microscopic level. Hydra can be prepared for

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transmission electron microscopy following different protocols (Holstein et al., 2010). Standard fixation with glutaraldehyde and osmium tetroxide followed by Epon embedding allows the visualization of SCs in Hydra spermatocytes (Fraune et al., 2012a). Immunolocalization analysis of SC proteins is usually performed on spread spermatocytes (and on oocytes). Similarly, only minor adaptions to the de Boer’s protocol for dry down spreading of mouse spermatocytes (de Boer et al., 2009) are sufficient to prepare adequate chromosome spreads of Hydra spermatocytes (Fraune et al., 2012a). Antibodies against purified HIS-tagged meiotic Hydra proteins can be applied to achieve staining of the target proteins following the standard protocol for immunolocalization on chromosome spreads (de Boer et al., 2009). Available antibodies detect the characterized SC components (Fraune et al., 2012a, 2013). They can also be used in double-label immunofluorescence experiments to detect components of the recombination machinery (Fig. 5). The lack of available cell culturing systems for mammalian meiotic cells led to the development of alternative strategies to investigate the properties of mammalian SC proteins in vitro. Transfections of somatic cells with transgenes encoding SC proteins allow studying their fate in the absence of any other meiotic protein (Fraune et al., 2012b). So far, these approaches revealed several binding and recruiting properties of the mammalian SC components in the artificial environment of transfected COS-7 cells. Mammalian SYCP1 and SYCP3 (Fig. 6B) can self-assemble to independent networks of higher ¨ llinger et al., 2005; Winkel order fibrils (Yuan et al., 1998; O et al., 2009) and therefore are described as bona fide structural SC components. Under the same experimental conditions, HySYCP3 is able to form aggregates that apparently can interact with the mammalian SYCP3 when co-transfected (Fig. 6A and C), resembling the co-assembly of mammalian and fish SYCP3 described earlier (Baier et al., 2007). Likely, the mammalian and cnidarian SYCP3 still have similar properties that allow them to co-assemble into higher order structures in the heterologous system despite of their sequence differences

Fig. 5. Immunolocalization of HySYCP3 and HyRPA on a chromosome spread preparation of Hydra spermatocyte. SYCP3 is a marker protein for the LEs of the SC (Fraune et al., 2012a), while RPA is marker protein for transition recombination nodules during the process of meiotic homologous recombination (Moens et al., 2007). Similar to the situation in mouse spermatocytes (Moens et al., 2007), RPA locates in several foci along the chromosome axes of early pachytene spermatocytes in Hydra (some of the chromosome axes are denoted by arrows). Scale bar, 10 mm.

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Fig. 6. Polymerization properties of HySYCP3 and rat SYCP3 in the heterologous system. Eukaryotic expression constructs of Hydra and rat Sycp3 were transfected into somatic COS-7 cells and detected in immunofluorescence analysis by the use of species-specific antibodies. HySYCP3 forms small aggregates in the cytoplasm (A and A0 ). When co-transfected with SYCP3 of the rat (RaSYCP3, C and C00 ), HySYCP3 (C0 ) is recruited into the fibrillar network (C000 ) that is characteristic for ectopic expressed vertebrate SYCP3 (B and B0 ). Scale bars, 20 mm.

Fig. 7. Polymerization properties of HySYCE2 and HyTex12 in the heterologous system. Eukaryotic expression constructs of wild type HySYCE2 and a myc-tagged HyTex12 were transfected individually (A and B) and combined (C) into somatic COS7 cells and detected by immunofluorescence microscopy with the aid of a-HySYCE2 and a-myc antibodies, respectively. Individually transfected HySYCE2 (A, A0 ) as well as myc-HyTex12 (B and B0 ) evenly diffuse in the cells. However, in co-transfection experiments HySYCE2 (C0 ) and myc-HyTex12 (C00 ) form large aggregates in the cytoplasm, indicating an interaction of the proteins (C000 ). Scale bars, 20 mm.

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that have accumulated over the last almost 600 million years. Similar to HySYCP3, HySYCP1 only assembles to aggregates when transfected into COS-7 cells. Extended networks as seen for mammalian SYCP1, however, were not detected. It remains to be clarified whether this finding represents a significant difference between the features of the mammalian and the cnidarian proteins since it cannot be excluded that culture conditions of COS-7 cells (i.e., 37 C), which differ from the optimal culturing temperature of Hydra (18 C), influence the polymerization properties. On the other hand, HySYCE2 and HyTex12, which distribute homogenously throughout the COS7 cells in individual transfections, co-assemble to elongated aggregates when co-transfected (Fig. 7). Remarkably, coassembly of higher order structures was reported previously in the case of their human homologues (Davies et al., 2012). In summary, expression studies as well as microscopic analysis and studies in the heterologous system can be easily applied to meiotic proteins of Hydra with only minor adaptions to standard protocols used in the mouse model. CONCLUSIONS The study of meiosis in Hydra revealed that this transparent fresh water polyp has a considerable inventory of SC protein components that show significant homology to the mammalian SC proteins. Though the differentiation pathway of the sperms in Hydra seems very similar to that in higher animals in morphological respects (Kuznetsov et al., 2001), nearly nothing is known about the conservation of molecular pathways and signaling in the germ line beyond the SC. As Hydra belongs to the most basal metazoan species, it represents an appealing and simple invertebrate system to complement the mammalian research of SC evolution, function and dynamics. It might be the right choice to further explore the evolutionary conservation of additional meiotic features. Testing of several methods that are commonly used in this field of research revealed that a large number of these can easily be adapted to the cnidarian system. The generation of transgenic animals for meiosis research, comparable to the established methods in the other model organisms, is the only exception to this. Transgenes for knock-down and specific overexpression of target proteins could be stably introduced into the Hydra genome of all three stem cell lineages. Constructs containing either a specific hairpin cassette fused to an egfp reporter gene or an egfp fusion gene, expressed under the control of a constitutively active promotor, were successfully microinjected into two- to eightcell-stage embryos as described (Wittlieb et al., 2006; Boehm et al. 2012; Franzenburg et al. 2012). Germ line transmission of transgenic constructs for the manipulation of meiotic genes, however, was not yet achieved reliably. Other methods for a gene silencing, for example the introduction of dsRNA by electroporation, have also been applied (Khalturin et al. 2008), but not tested for meiotic genes so far. A successful genetic manipulation of the germ line would be desirable to be reached in the near future since this approach together with the transparency of Hydra would allow to perform in vivo imaging of the dynamic behavior of fluorescently tagged SC proteins.

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ACKNOWLEDGEMENTS We thank Dr. Susanne Kramer and Dr. Jana Link (Biocenter, University of Wu¨rzburg, Germany) for their critical reading of the manuscript. This study was supported by a grant from the Priority Program of the German Science Foundation (SPP 1384, Mechanisms of Genome Haploidization).

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