Identification of new gene candidates on the sex chromosomes of the platyfishXiphophorus maculatus by Astrid Böhne (1), Christina Schultheis (2), Qingchun Zhou (2), Alexander Froschauer (2), Cornelia Schmidt (2), Yvonne Selz (2), Ingo Braasch (2), Catherine Ozouf-Costaz (3), Agnès Dettai (3), Béatrice Ségurens (4), Arnaud Couloux (4), Sylvie Bernard-Samain (4), Stefan Chilmonczyk (5), Alia Gannouni (1), Karim Madani (1), Frédéric Brunet (1), Delphine Galiana-Arnoux (1), Manfred Schartl (2) & Jean-Nicolas Volff (1)

AbsTract. - In order to better understand the molecular and evolutionary mechanisms driving sex determination in fish, bacterial artificial chromosome (BAC) contigs covering the sex determination region of the X and Y sex chromosomes of the platyfish Xiphophorus maculatus have been constructed. Initial analysis of these contigs led to the identification of eleven gene candidates closely linked to the master sex-determining locus and expressed in different types of tissues. These genes are all present on both the X and the Y chromosomes, suggesting a restricted degree of differentiation and probably a young evolutionary age for the platyfish sex chromosomes. Syntenies were detected with several zebrafish chromosomes as well as with two medaka and several tetrapod autosomes, supporting an independent origin of sex chromosomes not only in different vertebrate lineages but also in different fish species. Key words. - Sex chromosomes - Sex determination - Fish - Poeciliid - Xiphophorus.

Introduction sex determination (Volff and Schartl, 2001). This is particu- In contrast to the situation observed in mammals and larly true for the genus Xiphophorus, which is also an estab- birds, a huge diversity of sex determination mechanisms lished model for cancer research (Meierjohann et al., 2004). exists in fish (Devlin and Nagahama, 2002; Volff et al., In the platyfish X. maculatus, three types of homomorphic 2007). Closely related fish species can have different mecha- sex chromosomes have been identified: X, Y and W. Natural nisms of sex determination, reflecting a frequent switch females are XX, XW or ZW, whereas males are YY or XY. between sex determination systems during fish evolution. In WW females can be obtained under laboratory conditions. It order to better understand this phenomenon, interspecific is possible to derive strains with either male or female heter- comparative analyses of sex determination genes and sex ogamety and thus to study the link between different systems chromosomes are necessary. Major fish models under inves- within a same genus. Genetic and molecular markers are tigation include the Japanese medaka Oryzias latipes and available, including the Xmrk oncogene, which encodes a related species, the threespine stickleback Gasterosteus receptor tyrosine kinase inducing the formation of melano- aculeatus, the tilapias Oreochromis niloticus and O. aureus, ma. Two pigmentation loci are also linked to the master sex- the pufferfish Takifugu rubripes as well as several salmonids determining locus, as well as a polymorphic locus determin- (for review, Volff et al., 2007). So far, the master sex-deter- ing the onset of sexual maturity (Volff and Schartl, 2001). mining gene has been identified only in O. latipes (Matsuda As a first step towards the positional cloning of the mas- et al., 2002; Nanda et al., 2002). However, this gene, called ter sex-determining gene of the platyfish, a bacterial artificial dmrt1bY or dmy, is present in only a very restricted number chromosome (BAC) genomic library has been constructed of species and is therefore not the universal master sex- (Froschauer et al., 2002). We report here the identification of determining gene in fish. X- and Y-chromosomal genes through BAC clone sequenc- Poeciliids, a family of livebearers originally found in ing, which allowed establishing syntenic relationships Mexico and Central America, are very attractive organisms between the sex-determining region of X. maculatus and for the study of sex determination, sexual selection and many other fish and tetrapod genomes. other biological questions. Various types of sex-determining systems have been described between and even within spe- Methods cies, making this fish family extremely well-suited to deci- Fish pher the molecular and evolutionary basis of the diversity of X. maculatus (population Rio Jamapa, strains WLC1274

(1) institut de génomique fonctionnelle, Institut fédératif biosciences Gerland Lyon-Sud, Université de Lyon, 69003 France; INRA, CNRS, Université Lyon 1, École normale supérieure, 69364 France. [[email protected]] (2) University of Würzburg, Physiologische Chemie I, Biozentrum, Am Hubland, 97074 Würzburg, Germany. (3) Département systématique et évolution, CNRS UMR7138, Muséum national d'Histoire naturelle, 43 rue Cuvier, 75005 Paris, France. (4) Genoscope, 2 rue Gaston Crémieux, CP 5706, 91057 Evry c e d e x , France. (5) unité de virologie et immunologie moléculaires, Institut national de la recherche agronomique, Domaine de Vilvert, 78352 Jouy-en-Josas, France.

Cybium 2008, 32(2) suppl.: 69-71. Sex chromosomes in Xiphophorus Bö h n e e t a l . and WLC1235) was kept under standard conditions in aquar- reported (Volff et al., 2003; Schultheis et al., 2006); two ium facilities at the Biocenter of the University of Würzburg other genes, irf3 and KIAA1524, are described here for the (Germany) and the IFR128 BioSciences Gerland - Lyon Sud first time. As observed for other platyfish sex chromosomal in Lyon (France). These strains have a male heterogamety genes, irf3 and KIAA1524 are located on both the X and the system (females are XX and males XY). Y (Tab. I). However, the data available did not allow deter- mining with certainty if these genes are functional on both RT-PCR expression analysis types of sex chromosomes. Total RNA was extracted from adult tissues and organs Irf3, for interferon regulatory factor 3, encodes a phos- using the Total RNA Isolation Reagent (Abgene). PCR reac- phoprotein that, when activated, acts as a transcription factor tions were performed in 5% dimethyl sulfoxide (DMSO) on in the innate immune response and regulates interferon pro- cDNA prepared with the RevertAid First Strand cDNA Syn- duction in mammals. Binding to DNA takes place through thesis Kit (Fermentas Life Science). The amount of cDNA the IRF protein domain, also called tryptophan pentad repeat, was equalized according to RT-PCR for the actin gene (25 which is characteristic for the IRF family. Irf3 is activated cycles, annealing temperature 55°C) with primers XmacAct1 through viral and bacterial infections and possibly through (5’-GTAGGTGATGAAGCCCAGAGC-3’) and XmacAct2 cellular stress signals (Grandvaux et al., 2002). (5’-AGGGAGCTCGTAGCTCTTCTC-3’) (not shown). KIAA1524 encodes a protein also called p90 (“90kDa PCR for irf3 (35 cycles) was performed at an annealing tem- companion auto-antigen of p62”; Soo Hoo et al., 2002). Like perature of 65°C with primers IRF_W2_F2 5’-AGCT- p62, a cytoplasmic RNA-binding protein regulating the GGCGAACGGAGAGAAG-3’ and IRF_W2_R2 5’-ACAG- expression of the insulin-like growth factor II mRNA, p90 is CAGCACCGACTCCATG-3’. PCR for KIAA1524/p90 (35 overexpressed in different types of cancer and recognized by cycles) was done at an annealing temperature of 57°C with auto-antibodies. Nothing is known so far concerning the primers P90F2 5’-TCAACATCATCTTGAACGGC-3’ and function of this protein. P90 contains a coiled-coil region P90R1 5’-GAAGTCTGGAAGGGGCGTGG-3’. (Soo Hoo et al., 2002) as well as moderately conserved Sequence analysis domains suggesting a role either as a chromosome segrega- BAC clones were shotgun-sequenced at the Genoscope tion ATPase (cluster of orthologous group COG1196.2) RT- center (http://www.cns.fr/). Gene candidates were identified PCR expression analysis of the sex chromosomal genes irf3 through sequence comparison with public databases (http:// (interferon regulatory factor 3) and KIAA1524 (aka p90 www.ncbi.nlm.nih.gov/BLAST/; BLASTX). Localization of autoantigen) or as an autophagy protein involved in intracel- orthologous genes in other vertebrates was investigated lular degradation in autophagosomes (protein family through BLAST analysis of genome assemblies (http://www. pfam08614.1). irf3 KIAA1524 ncbi.nlm.nih.gov/BLAST/; http://www.ensembl.org/Multi/ Expression of both and was detected by RT-PCR in both male and female adult gonads, as well as in blastview). all other types of tissues and organs tested (Fig. 1). This is Results and discussion similar to the broad expression profile in mammals identified Initial sequencing and analysis of BAC clones covering through expressed sequence tag analysis (data not shown). the sex-determining region of the platyfish uncovered eleven None of the genes displayed a clear sex-specific expression gene candidates (Tab. I). Some of them have already been suggesting a function in sex determination, as reported for

Table I. - Gene candidates on the sex chromosomes of the platyfish and their localization in other vertebrate genomes. Dre, Danio rerio; EGF, epidermal growth factor; Gga, Gallus gallus; Hsa, Homo sapiens; Mmu, Mus musculus (mouse); Ola, Oryzias latipes; Scaf, scaf- folds.

70 Cybium 2008, 32(2) suppl. Bö h n e e t a l . Sex chromosomes in Xiphophorus

basis of sex-linked differences in resistance to virus infec- tion, disease and environmental stress. Acknowledgements. - This work is funded by grants from the Deutsche Forschungsgemeinschaft (to M.S. and J.-N.V.), the Bio- Future programme of the Bundesministerium für Bildung und For- schung, the Association pour la recherche contre le cancer, the Institut national de la recherche agronomique, the Fondation pour la recherche médicale and the Centre national de la recherche sci- Figure 1. - RT-PCR expression analysis of the sex chromosomal genes irf3 (interferon regulatory factor 3) and KIAA1524 (aka p90 entifique (to J.-N.V.) and the Fonds der Chemischen Industrie (to autoantigen) in platyfish adult tissues and organs. M. S.). References the medaka sex-determining gene (Matsuda et al., 2002; DEVLIN R. & Y. NAGAHAMA, 2002. - Sex determination and Nanda et al., 2002). However, a stronger expression of sex differentiation in fish: An overview of genetic, physiologi- KIAA1524 was noted in testis compared to ovary and other cal and environmental influences. Aquaculture., 208: 191-364. organs (Fig. 1). FROSCHAUER A., KÖRTING C., KATAGIRI T., AOKI T., ASA- KAWA S., SHIMIZU N., SCHARTL M. & J.-N. VOLFF, 2002. The localization of sequences orthologous to platyfish - Construction and initial analysis of bacterial artificial chromo- genes in other vertebrates allowed establishing syntenic rela- some (BAC) contigs from the sex-determining region of the tionships of the sex-determining region of X. maculatus with platyfish Xiphophorus maculatus. Gene, 295: 247-254. human chromosome 2 and five other autosomes, as well as GRANDVAUX N., SERVANT M.J., TENOEVER B., SEN G.C., BALACHANDRAN S., BARBER G.N., LIN R. & J. HIS- with seven mouse and four chicken autosomes (Tab. I). The COTT, 2002. - Transcriptional profiling of interferon regulatory gene order was found to be conserved over four zebrafish factor 3 target genes: Direct involvement in the regulation of chromosomes and only two medaka autosomes, reflecting interferon-stimulated genes. J. Virol., 76: 5532-5539. the closer phylogenetic relationship between O. latipes and MATSUDA M., NAGAHAMA Y., SHINOMIYA A., SATO T., X. maculatus. No synteny was found with any sex chromo- MATSUDA C., KOBAYASHI T., MORREY C.E., SHIBATA N., ASAKAWA S., SHIMIZU N., HORI H., HAMAGUCHI S. some of any species, supporting an independent origin of & M. SAKAIZUMI, 2002. - DMY is a Y-specific DM-domain gonosomes not only in different vertebrate lineages but also gene required for male development in the medaka fish. Nature, between different fish species. 417: 559-563. MEIERJOHANN S., SCHARTL M. & J.-N. VOLFF, 2004. - Conclusions Genetic, biochemical and evolutionary facets of Xmrk-induced All gene candidates identified so far on the sex chromo- melanoma formation in the fish Xiphophorus. Comp. Biochem. somes of the platyfish have been detected on both the X and Physiol. C Toxicol. Pharmacol., 138: 281-289. the Y, reflecting the poor degree of differentiation of the SCHULTHEIS C., ZHOU Q., FROSCHAUER A., NANDA I., SELZ Y., SCHMIDT C., MATSCHL S., WENNING M., gonosomes and their probable recent origin. Even if we can VEITH A.-M., NACIRI M., HANEL R., BRAASCH I., DET- not exclude that some genes are functional only on one type TAI A., BÖHNE A., OZOUF-COSTAZ C., COULOUX A., of sex chromosome, this contrasts with the situation observed BERNARD-SAMAIN S., SCHMID M., SCHARTL M. & J.-N. for the master sex-determining genes in medaka (dmrt1bY) VOLFF, 2006. - Molecular analysis of the sex-determining region of the platyfish Xiphophorus maculatus. Zebrafish, 3: and mammals (Sry), which are specific of one type of sex 295-305. chromosomes (the Y). In addition, both irf3 and KIAA1524 SOO HOO L., ZHANG J.Y., & E.K. CHAN, 2002. - Cloning and are expressed in both male and female gonads as well as in characterization of a novel 90 kDa ‘companion’ auto-antigen of other organs. Hence, none of the genes identified does repre- p62 overexpressed in cancer. Oncogene, 21: 5006-5015. sent an obvious candidate for the master sex-determining VOLFF J.-N. & M. SCHARTL, 2001. - Variability of genetic sex determination in poeciliid fishes. Genetica, 111: 101-110. gene of the platyfish, or for any other linked locus. However, VOLFF J.-N., KÖRTING C., FROSCHAUER A., ZHOU Q., the possible biological relevance of the higher expression of WILDE B., SCHULTHEIS C., SELZ Y., SWEENEY K., KIAA1524 in male gonads as well as the function of the pro- DUSCHL J., WICHERT K., ALTSCHMIED J. & M. tein remains to be investigated. Finally, the embryonic SCHARTL, 2003. - The Xmrk oncogene can escape nonfunc- expression of these genes, which might be different from the tionalization in a highly unstable subtelomeric region of the genome of the fish Xiphophorus. Genomics, 82: 470-479. adult pattern, still needs to be examined. Finally, the pres- VOLFF J.-N., NANDA I., SCHMID M. & M. SCHARTL, 2007. - ence of a key immune response gene like irf3 close to the Governing sex determination in fish: Regulatory putsches and sex-determining locus might allow studying the molecular ephemeral dictators. Sex. Dev., 1: 85-99.

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