| Unlike many basic cellular processes shared across vast phylogenetic distances, sex-development pathways are highly variable between phyla. Despite morphologically distinct males and females are observed throughout the animal kingdom, Sex-determining mechanisms differ completely between phyla, including X- and Y-chromosome heterogametes in male mammals, Z- and W-chromosome heterogametes in female birds, and a temperature dependent sex determination in reptiles, etc. It is inferred that sex-determining mechanisms evolve rapidly, possibly more than other important developmental processes. But another hypothesis is proposed by Marin and Baker, sex determination mechanisms do not change more rapidly than other developmental processes, but rather that diversity in sex determination has been easier to identify. In any case, these varieties provide an excellent opportunity to study how the regulatory networks change, it also complicates the search for their origins.A hypothesis was put forward proposing that the genes involved in sex development process evolved rapidly and the most downstream genes in the cascade could be conserved, this predication has been supported by finding of doublesex-related genes which are widely conserved and involved in sexual development among phyla, e.g. doublesex (dsx) of Drosophila, mab-3 of Caenorhabditis elegans and DmrtllDMY of vertebrates. These DM domain genes, doublesex (dsx) (Drosophila), mab-3 (C. elegans) and Dmrtl (vertebrates), are the only similar molecule found in sexual development of metazoans from nematode, fly through to mammals. All these related genes have a common DNA-binding domain, termed the DM domain, which encode putative transcription factors. Both DSX and MAB-3 control the differentiation of sex specific structures and arefunctionally related in both Drosophila and C. elegans. The Dmrtl retains still its characteristic in roles of sexual development in evolution. Human DM gene, DMRT1, is located on the short arm of chromosome 9. Deletions of this region of 9p24 are associated with XY sex reversal. Male-specific expression for the gene in early gonadogenesis is consistent with its role in testis development. In mouse, higher Dmrtl expression is necessary for testicular differentiation, whereas lower expression is compatible with ovarian differentiation. A Dmrtl knockout mouse demonstrated that the gene is required for testis differentiation after determination, but dispensable for ovary development. In several other vertebrates, Dmrtl genes have also been identified. In chicken, the Dmrtl is sex-linked, on the Z chromosome, and there is a higher dosage of the gene in the male as compared with the female. The homologs of the Dmrtl have also been cloned in turtle and alligator. The expression of Dmrtl was found to be sexually dimorphic in reptile species, and was stronger in embryos developing under male-specific temperature than in female ones. And the autosomal Dmrtl gene may have transposed to the Y chromosome and become a master regulator gene (Dmrtly/DMY) in male determination in some fish (medaka). These data show that the DM genes from invertebrates to vertebrates are sex-specific, often associated with male-specific development.The DM domain gene family has multiple members in both invertebrates and vertebrates. For example, Up to date, there are at least seven DM domain genes found in mouse, eight in human, four in Drosophila, eleven in C. elegans, and six in fish (Dmrt 1-5 and Dmrt2b), all of them encode putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of C. elegans. Multiple DM domain genes have been suggested to be involved in mouse sexual development. At least three murine DM domain genes in addition to Dmrtl are expressed in the embryonic gonad, including Dmrt3, Dmrt4, and Dmrtl. However, Dmrt2 is expressed in presomitic mesoderm and developing somites, while DmrtS and Dmrt6 are expressed primarily in the brain, suggesting a role in other developing processes.Although some of the DM genes are involved in sexual development, function of most of these genes remains unclear, and we know remarkably little about the evolution ofthe DM genes. It is still the question that the male-specific role of the DM genes is primordial during their evolution, or independently evolved by convergence. Furthermore, it is essential to understand the roles of the DM genes in regulatory pathway of sex determination, or if any, the roles in other developmental processes. There is also few structural and functional analysis concerning DM domain genes of the model fish, zebrafish, except terra/Dmrt2a, which was shown involving in process of somite development other than the sexual development. We report here cloning, characterization and expression of Dmrtl and Dmrt5 of zebrafish.In zebrafish, we identified five new Dmrt members by degenerate PCR amplification of DM domains, including Dmrtl, Dmrt2b, Dmrt3, Dmrt4 and Dmrt5. Zebrafish Dmrtl, Dmrt2, and Dmrt3 genes were shown to form a gene cluster, which was located on chromosome linkage group5 (LG5) of zebrafish. The synteny of these Dmrt genes was conserved in human, mouse, rat, fugu and medaka. The other DM domain genes in zebrafish are still waiting for identification.Based on the DNA sequence information, we designed primers for cloning full length Dmrtl. The cDNAs synthesized from SMART primer were used as template for both 5' RACE and 3' RACE by combined use of the primer pairs of DM domain and SMART primer. After both 5' and 3' half regions of Dmrtl sequence, which overlap in DM domain region, were obtained, full length Dmrtl sequence was amplified by PCR based on the 5' and 3' sequence information. Interestingly, there are at least three kinds of alternative splicing events in zebrafish gonads, which all occurred in the 3' region after the DM domain. Dmrtl al were a 138Obp full length cDNA encoding a predicted protein with 267 amino acids. In Dmrtl b, a deletion of the nucleotides CACACACA at nt 710 results in a frame shift with a different ORF of 242 or 254 amino acids (Dmrtl bl and Dmrtl b2). Dmrtl cl codes a potential protein with 132 amino acids. To get more information about the genomic structure of the Dmrtl, we searched the zebrafish (Danio rerio) Assembly database (Sanger Institute), and found the contig encoding the Dmrtl cDNAs. After compared the genomic sequence with mRNA sequences, we found that there were seven exons, which were alternatively spliced to generate the Dmrtl isoforms.The Drosophila Dsx gene controls somatic sexual differentiation by producing alternatively-spliced mRNAs with different 3' region encoding related sex-specific protein DSXm in males and DSXf in females. Recently, we first observed the similar Dmrtl 3' splicing pattern with that of Drosophila Dsx, which generated three isoforms in gonads of the rice field eel, a fresh-water fish with sex transformation characteristic from female to male during its life. The alternative splicing was also observed in the Dmrtl of coral (Acropora millepora) and lizard {calotos versicolar). It appears likely that the alternatively splicing pattern of DM genes is evolutionarily conserved.All these zebrafish Dmrtl protein sequences were aligned with Drosophila DSX, C. elegans MAB-3, Dmrtl and DMY of the other fishes, turtle, frog, birds and mammals, which show conserved DM domain and diverse 3' and 5' regions. Another two conserved domains (domain I and II) in the 3' region were observed in both Dmrtl a and b, but not in Dmrtl c of zebrafish. We further constructed a phylogenetic tree based on amino acid sequences from all these DM proteins. These results show that the DM gene of zebrafish is clustered into the Dmrtl gene group of the other animals, and the DM proteins are evolutionarily conserved. An alignment of intron-exon junctions of Dmrtl of different species shows that intron-exon junctions of Dmrtl were conserved among phyla.To analyze the expression patterns of these spliced Dmrtls, we assayed their expression by RT-PCR using RNA isolated from adult gonads. All isoforms of the Dmrtls expression are detectable by RT-PCR in both male and female gonads of zebrafish. Quantitative RT-PCR analysis and Northern blotting further indicated that Dmrtl relative expression level in testis is higher than ovary. To determine expression region in gonads of zebrafish, we performed Dmrtl mRNA in situ hybridization to gonad sections. In zebrafish, expression pattern of the Dmrtl is quite different. Expression signals were observed in developing germ cells.Using same methods, we cloned the zebrafish DmrtS with full length of 1419bp, which consists of two exons, and encodes a 440-amino acid protein with conserved DMA and DMB domains in addition to DM domain. Phylogenetic analysis shows that zebrafishDmrtS fits within the Dmrt5 clade of fish and mammals. Genome data do not show the exact information of location of DmrtS gene now, but data from pre-emsembl indicated that DmrtS gene maybe localized in LG8.Reverse transcription polymerase chain reaction (RT-PCR) analysis of total RNAs of different stages of embryos showed that DmrtS transcript appeared in early gastrula period, subsequently increased to a high level in late stage of gastrula period (bud stage) and lower until the hatch period. Quantitative RT-PCR analysis further indicated that relative expression level reached peak at stage 26-somites. Whole mount in situ hybridization was further used to analyze expression sites in embryos, which revealed that expression of zebrafish Dmrt5 was showed in central nervous system, specially in mid brain and mid-hind brain boundary.Reverse transcription polymerase chain reaction analysis of adult tissues showed that zebrafish DmrtS was specially expressed in brain, testis and ovary, but not in other tissues such as muscles, liver and intestine. Real time fluorescent quantitative RT-PCR was further used to determine expression level, which showed that expression is higher in testis than ovary. Clearly, this pattern of zebrafish DmrtS was quite distinct from that of human DMRTS. Expression of human DMRTS was only detected in adult testis. Although mouse DmrtS expression profiles of adult is not available, in 14.5 dpc embryos, it was expressed mainly in brain, testis and ovary, and expression were also detected in several other tissues. To examine expression sites in gonads of zebrafish Dmrt5, we further performed in situ hybridization to gonadal sections. Antisense probe for DmrtS showed expression in developing germ cells, especially in spermatogonia, spermatocytes, spermatids and sperm cells, and in ovary, its expression signals appeared in developing oocytes.
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