Ute Hornung

A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes

The genes that determine the development of the male or female sex are known in Caenorhabditis elegans, Drosophila, and most mammals. In many other organisms the existence of sex-determining factors has been shown by genetic evidence but the genes are unknown. We have found that in the fish medaka the Y chromosome-specific region spans only about 280 kb. It contains a duplicated copy of the autosomal DMRT1 gene, named DMRT1Y. This is the only functional gene in this chromosome segment and maps precisely to the male sex-determining locus. The gene is expressed during male embryonic and larval development and in the Sertoli cells of the adult testes. These features make DMRT1Y a candidate for the medaka male sex-determining gene.

Evolutionary Origin of the Medaka Y Chromosome

Genetic sex determination in an XX-XY chromosome system can be realized through a locus on the Y chromosome that makes the undifferentiated gonad develop into a testis. Although this mechanism is widespread, only in two cases so far have the corresponding master male sex-determining genes been identified. One is Sry, which initiates testes determination in most mammals. The other is dmrt1bY (syn. dmy), from the fish medaka, Oryzias latipes. The mammalian Y is roughly estimated to be over 200 million years old. The medaka Y may be considerably younger. A comparative analysis of the genus Oryzias revealed that one sister species of the medaka has dmrt1bY on a homologous Y chromosome, whereas in another closely related species only a non-sex-linked pseudogene is present. In all other species, dmrt1bY was not detected. The divergence time for the different species was determined with mitochondrial DNA sequences. The timing was confirmed by independent calculations based on dmrt1 sequences. We show that the medaka sex-determining gene originated approximately 10 million years ago. This makes dmrt1bY and the corresponding Y chromosome the youngest male sex-determining system, at least in vertebrates, known so far.

Molecular cloning and characterization of DMRT genes from the medaka Oryzias latipes and the platyfish Xiphophorus maculatus

The DMRT genes constitute a family of genes, which possess a common motif called the DM domain. DMRT1 is considered to be involved in sex determination and/or sex differentiation, but not much information exists about the function of the other gene family members. We cloned DMRT genes of two important model fish species, the medaka, Oryzias latipes, and the platyfish, Xiphophorus maculatus. Based on sequence similarity and genomic structure with known DMRT genes, the gene from the medaka was identified as OlaDMRT4, and those from the platyfish as XmaDMRT2 and XmaDMRT4. OlaDMRT4 was assigned to the linkage group 18 (LG18) of the medaka by linkage analysis and fluorescence in situ hybridization. The earlier cloned medaka DMRT1, 2 and 3 genes form a cluster on LG9. Therefore, OlaDMRT4 does not belong to the DMRT gene cluster. In adult medaka fish, OlaDMRT4 is expressed in the brain, eyes, gill, kidney, as well as testis and ovary. During development, OlaDMRT4 exists as maternal transcripts, and is expressed until early larval stages. This pattern of expression differs from the other known medaka DMRT genes. Surprisingly it is also not the same as its putative tilapia ortholog (DMO). These differences in expression suggest that DMRT4 might fulfill divergent functions in different species.

Developmentally regulated and non-sex-specific expression of autosomal dmrt genes in embryos of the Medaka fish (Oryzias latipes)

The dmrtgene family of vertebrates comprises several transcription factors that share a highly conserved DNA-binding domain, the DM domain. Like some of their invertebrate counterparts, e.g. Drosophila doublesex (dsx) and the Caenorhabditis elegans Mab3, several are implicated in sex determination and differentiation. Thus far, dmrt genes represent the only factors involved in sexual development that are conserved across phyla. In the teleost Medaka (Oryzias latipes), a duplicated copy of dmrt1, designated dmrt1bY or dmy, has recently been postulated to be the master regulator of male development in this species. Here, we have analyzed the expression of four additional Medaka dmrt genes during embryonic and larval development. In contrast to other vertebrates, the autosomally located dmrt1a gene of Medaka is not expressed at detectable levels during embryogenesis. On the other hand, dmrt2, dmrt3 and dmrt4 show highly restricted and non-overlapping expression patterns during embryogenesis. While dmrt2 is expressed in early somites, dmrt3 transcripts are found in dorsal interneurons and dmrt4 is expressed in the developing olfactory system. Other than in mouse, they do not show any sex specific expression and no transcription could be detected in the early developing gonads. However, all four analyzed dmrt genes share expression in the differentiating gonad of larvae and in adult testis.

Fish retroposons related to the Penelope element of Drosophila virilis define a new group of retrotransposable elements

Abstract.Poseidon and Neptune are two ancient lineages of retroposons related to the Penelope element from Drosophila virilis. They have been identified in various teleost fish species, including the medakafish (Oryzias latipes), and the pufferfishes Fugu rubripes and Tetraodon nigroviridis, whose genomes are currently being sequenced. Some of these elements are highly reiterated in fish genomes. Penelope-related elements were also identified in blood fluke, shrimp, sea urchin, cichlid fish and frog, showing that they are widespread in animals. Penelope-related retroposons were not detected among sequences from the Drosophila melanogaster and human genome projects, suggesting that they have been lost from certain animal lineages. A sequence encoding a putative Uri (also called GIY-YIG) endonuclease domain was detected downstream from the gene for reverse transcriptase. To the best of our knowledge, this type of endonuclease sequence has previously been identified in group I introns and in genes for prokaryotic excinucleases but not in retrotransposable elements. Penelope-related elements are frequently truncated at their 5′ ends and can also be flanked by long terminal repeat-like structures. Phylogenetic analysis of the reverse transcriptase domain failed to assign Penelope-related retroposons to one of the major groups of retroelements. Overall, therefore, the evidence strongly suggests that these sequences represent a new group of retrotransposable elements.

Inhibition of primordial germ cell proliferation by the medaka male determining gene Dmrt1bY

BackgroundDmrt1 is a highly conserved gene involved in the determination and early differentiation phase of the primordial gonad in vertebrates. In the fish medaka dmrt1bY, a functional duplicate of the autosomal dmrt1a gene on the Y-chromosome, has been shown to be the master regulator of male gonadal development, comparable to Sry in mammals. In males mRNA and protein expression was observed before morphological sex differentiation in the somatic cells surrounding primordial germ cells (PGCs) of the gonadal anlage and later on exclusively in Sertoli cells. This suggested a role for dmrt1bY during male gonad and germ cell development.ResultsWe provide functional evidence that expression of dmrt1bY leads to negative regulation of PGC proliferation. Flow cytometric measurements revealed a G2 arrest of dmrt1bY expressing cells. Interestingly, also non-transfected cells displayed a significantly lower fraction of proliferating cells, pointing to a possible non-cell autonomous action of dmrt1bY. Injection of antisense morpholinos led to an increase of PGCs in genetically male embryos due to loss of proliferation inhibition.ConclusionIn medaka, dmrt1bY mediates a mitotic arrest of PGCs in males prior to testes differentiation at the sex determination stage. This occurs possibly via a cross-talk of Sertoli cells and PGCs.

Hormonal Induction and Stability of Monosex Populations in the Medaka (Oryzias latipes): Expression of Sex-Specific Marker Genes

Abstract The model teleost medaka (Oryzias latipes, d-rR.YHNI strain) was used to produce offspring of a defined sex (monosex populations) by crossing experimentally produced YY and XX males to normal females. These monosex populations had the predicted chromosomal constitution as shown by a sex chromosome-specific DNA sequence. However, in XX populations the spontaneous development of males without previous exposure to androgens was observed. Differences in the percentage of male offspring from individual XX breeding pairs indicate a possible variation of unknown genetic factors to be responsible for the development of XX males. The expression of two gonadal genes that are involved in sex differentiation, Dmrt1b(Y) and Fig1a (factor in the germ line α), was analyzed in monosex populations. Dmrt1b(Y) expression correlated strictly with the genotype but not the sexual phenotype. When XY juvenile fish were exposed to 17α-ethynylestradiol at concentrations that induce sex reversal, Dmrt1b(Y) expression was not repressed. However, Dmrt1b(Y) was expressed in XY or YY gonads regardless of the sex and could not be detected in XX individuals. In contrast, the expression of Fig1a correlated with the phenotypic sex: Fig1a was expressed in male juvenile fish exposed to 17α-ethynylestradiol and repressed in fish exposed to 17α-methyltestosterone. The Dmrt1b(Y) expression appears to reflect an early and important event in sex determination and lends support to the suggested key regulatory role of the Dmrt1b(Y) gene in sex determination. This process is apparently hormone insensitive, and the expression of further downstream acting genes can be regulated (directly or indirectly) by sex steroids.

Expression of the Male Determining Gene dmrt1bY and Its Autosomal Coorthologue dmrt1a in Medaka

Dmrt1bY (aka dmy) has been identified as the master male sex-determining gene of medaka by positional cloning and genetic evidence. How this gene on the molecular level determines the differentiation of the gonad primordium towards a testis, however, is unknown. To better understand the roles of dmrt1bY and its autosomal counterpart dmrt1a in the process of sex determination, a detailed expression analysis by quantitative real-time RT-PCR was performed showing non-organ-specific expression of dmrt1bY during embryogenesis and low expression in adult testes. Surprisingly a high expression of dmrt1bY was noted in the spleen of males. Dmrt1a is exclusively expressed in adult testes, 50 times higher than dmrt1bY. Dmrt1bY mRNA and protein have short half-lives and the GFP fusion protein products of both dmrt1 genes are localized to the nucleus. These features are consistent with the suggestion that Dmrt1 proteins might function as DNA-binding transcription factors.

TRIPLET REPEAT VARIABILITY IN THE SIGNAL PEPTIDE SEQUENCE OF THE XMRK RECEPTOR TYROSINE KINASE GENE IN XIPHOPHORUS FISH

Trinucleotide repeats in several human genes have been found to undergo spontaneous variation in repeat numbers in succeeding generations. Expansion of the repeat beyond a certain length causes specific pathological disorders. So far, a naturally occurring triplet repeat instability of transcribed sequences has been reported only from humans. However, the signal peptide encoding region of the receptor tyrosine kinase gene Xmrk from fish of the genus Xiphophorus contains a CTG repeat that differs in length even between closely related individuals. The consequence of this variability is signal peptides with shorter or longer hydrophobic core regions reaching, in some individuals, the critical maximum length for functional protein export or even exceeding it. In one stock, animals that are homozygous for such an allele were extremely rare, indicating that the triplet repeat length variability of the Xmrk gene of Xiphophorus may indeed have an influence on the function of the gene product and, under certain conditions, may affect the fitness of the individual.

DMRT Genes and Sex Determination in Medaka

The vast majority of animal species are bisexual, and in many cases the decision, if an embryo develops to a male or female, is made by the genome. Sex determination genes initiate a series of developmental processes that establish the male or female phenotype. The genetic scenarios how sex can be determined are of extraordinary diversity and have provoked questions on the evolution and maintenance of bisexuality. One of the unsolved problem is, why such a simple and highly conserved biological phenomenon as the existence of males and females should be controlled by so different mechanisms like the ratio of X chromosomes to autosomes for instance in the fruit fly Drosophila melanogaster and the worm Caenorhabditis elegans or the presence of a Y chromosome in male mammals.