The Bioinformatical Analysis On DMRT Family And Possible Role Of Dmrt4/Dmrt6in The Development Of Tilapia Gonad

DMRT (Doublesex and Mab-3related transcription factors) gene family has been proved to participate in a variety of processes, including growth and development, illness, especially in sex determination and differentiation. So far, about40members were identified in vertebrates and invertebrates. A conservative DM domain which could recognize specific DNA sequences is the main characteristics of the members. Bioinformatical analysis on DMRT family found that DMRT gene has existed in multicellular life such as coelenterate coral and related to sexual reproduction. Existing studies of DMRT genes on sex determination and differentiation were mainly focus on Dmrtl. To explore the potential role of other DMRT genes on sex determination and differentiation, gonadal development, we isolated all DMRT gene family members of Nile tilapia(Oreochromis niloticus), analyzed their gene structure, synteny and phylogenetic relationship and expression profile in gonadal ranscriptomes, and explored the possible role of Dmrt4/Dmrt6in the development of gonads by TALEN(Transcription activator-like effector nucleases).We isolated7DMRT genes in Nile tilapia via analysis of gonadal transcriptome and genome sequences in the present study. They are Dmrtl, Dmrt2a, Dmrt2b, Dmrt3, Dmrt4, Dmrt5and Dmrt6. Dmrt2a and Dmrt2b diverge from the replication of Dmrt2, and we couldn’t find other Dmrt2b homology in tetrapods. We speculate that duplication of Dmrt2may be unique to teleosts. Dmrt6was found to exist in teleost genome for the first time except tetrapods, and right now it was only isolated from cichlids by bioinformatical analysis. There are two possible reasons for this,1) secondary lost of Dmrt6in other fishes;2) incomplete sequencing of the fish genome, which was not including Dmrt6. Alignment of tilapia and tetrapods Dmrt6demonstrated that the similarity between tilapia Dmrt6and its mammalian counterparts was higher than60%. Phylogenetic and syntenic analyses indicated that tilpia DMRT genes were the homologous genes of their tetrapod counterparts. Five DMRT genes were found to be expressed in gonads by analysis of8gonad transcriptomes. The expression of Dmrtl and Dmrt6was higher in testes than ovaries, while the expression of Dmrt2a and Dmrt4was higher in ovaries than testes.RT-PCR, Real-time PCR, ISH (in situ hybridzation) and IHC (immunohistochemistry) were used to analyze the expression of tilapia Dmrt4/Dmrt6. Tissue distribution analysis indicated that Dmrt4was mainly expressed in the ovary, very low in the gill and muscle; while Dmrt6was primarily in the testis and low in other tissues, such as brain, pituitary, spleen, etc. Real-time PCR demonstrated that Dmrt4and Dmrt6was higly expressed from70dah(days after hatching) XX ovaries and90dah XY testes to adult, respectively, during normal development. Dmrt4expression was down-regulated during Fadrozole induced XX female to male sex reversal, while it was up-regulated during E2(estradiol) induced XY male to female sex reversal. In contrast, Dmrt6expression was up-regulated during Fdrozole induced XX female to male sex reversal, while it was down-regulated during E2induced XY male to female sex reversal. These results suggested that the expression of tilapia Dmrt4and Dmrt6were phenotype-related. ISH and IHC showed that tilapia Dmrt4was expressed in oocytes, while Dmrt6in spermatocytes. Promoter analysis demonstrated that tilapia Dmrt6can repress the expression of Cypl9ala, but Dmrt4can not. In order to study the role of tilapia Dmrt6during the gonadal development, TALEN were used to knock out Dmrt6. The results showed that TALEN could efficiently target Dmrt6. Histological observations of the3-month-old Dmrt6deficiency testes revealed that they developed abnormally with less spermatocytes and not well developed seminiferous ducts. Immunohistochemistry analysis showed that the expression of Cypl1b2and Dmrtl in Dmrt6deficiency testes was lower than those in control testes. No difference in Amh expression was detected in Dmrt6deficiency and control testes.In summary, we identified7DMRT genes in Nile tilapia genome, Dmrt6was first found in teleosts except tetrapods, bioinformatical analysis indicated that tilapia Dmrt6was the homolog of mammalian Dmrt6. Five DMRT genes were found to be expressed in gonads by transcriptome analysis. They may play important roles in sex determination and differentiation, and gonadal development. ISH and IHC analysis showed that tilapia Dmrt4was mainly expressed in oocytes, while Dmrt6mainly in spermatocytes. Their expression was phenotype-related. Promoter analysis indicated that Dmrt6can repress the expression of Cypl9ala, but Dmrt4can not. Knock out Dmrt6by TALEN demonstrated that Dmrt6deficiency resulted in less spermatocytes and seminiferous ducts, and therefore, it may be involved in spermatogenesis and development of seminiferous ducts.