Sex is used as a mode of reproduction in almost all vertebrate lineages. However, the signals determining if an embryo will develop as a male or a female are generally not conserved between major taxa. Even more surprising are the dramatic differences in evolutionary dynamics observed for sex determination between different groups of vertebrates. For example, mammals and birds have different systems of genetic sex determination, but both systems have been stably maintained over at least 100 million years of evolution in their respective lineages. In contrast, sex determination is extremely variable and evolves very rapidly in fish. Beside different forms of hermaphroditism, almost all possible forms of genetic sex determination have been observed, from male and female heterogamety with or without influence of autosomal loci to more complicated systems involving several loci but without sex chromosomes (polyfactorial sex determination), or several sex chromosomes and even several pairs of sex chromosomes. In numerous species, sex determination can be influenced by temperature and other environmental factors like the pH of the water or even social parameters. In most species, phenotypic sex can be fully reversed by hormone treatment. Importantly, a switch between sex determination systems is frequently observed during evolution in fish. In order to better understand the molecular and evolutionary basis of the diversity of sex determination in fish, the master sex-determining gene(s) -i.e. the gene(s) deciding the sex of individuals, like SRY in mammals- have to be identified in several fish species. So far, the master sex-determining gene has been reported only in the medaka Oryzias latipes. However, this gene, called dmrt1bY, is present in only a very restricted number of species and is therefore not the universal sex-determining gene in fish. Alternative models like the threespine stickleback, tilapia, salmonids and 'our' platyfish are necessary to identify and compare master sex-determining genes and better understand the evolutionary dynamics of sex determination in fish. We have initiated the positional cloning of the sex-determining gene of the platyfish Xiphophorus maculatus, an aquarium fish with many genetic and molecular markers available for the sex chromosomes. A bacterial artificial chromosome (BAC) library of XY males has been constructed and BAC contigs covering about 3 megabases of both X and Y chromosomes have been assembled. Fifty BAC clones covering the sex-determining region have been sequenced and are being assembled and analysed. About 60 sex chromosomal genes have been already identified, ten of them being also present on the mammalian X chromosome. Several new genes not described so far in any organism and with putative functions in sex determination and sex differentiation are particularly under scrutiny. Two ancient gene duplicates organized in tandem are expressed specifically in gonads not only in platyfish but also in medaka and even in Xenopus. Another gene, predominantly expressed in testis, is present at multiple copies of both the X and Y chromosomes of the platyfish. Finally, a last gene called Swimy is present on the Y chromosome in the sex determination region but not on the X, as expected for a male sex-determining gene. While a shorter form of Swimy with putative DNA- and protein interaction domains is expressed almost ubiquitously, a longer form including an additional alternative exon is expressed exclusively in testis. This male-specific protein contains domains potentially involved in posttranslational protein modifications mediated by the peptide SUMO-1. In this project, we propose (i) to finish the analysis of the BAC contigs and the comparison between X and Y chromosomes, with filling in of remaining gaps (ii) to systematically analyse the expression in embryos and adults of sex chromosomal genes not tested so far though RT-PCR and in situ hybridization (iii) to analyse at the functional level through mutant analysis, ectopic expression and knock-down technology the best candidates for a function in sexual development and (iv) to analyse the evolutionary dynamics of sex chromosomes and sex chromosomal genes identified in platyfish, particularly in the context of other systems of sex determination in other species. This project, involving national and international collaborations, will reveal new insights into the evolutionary dynamics of sex chromosomes and sex determination, with the identification a new gene able to control vertebrate sex determination. This study has implications for the aquaculture, where molecular sexing and manipulation of sex determination represent important economical challenges for the control of fish reproduction and growth.