Identification Of MicroRNAs In Medaka Gonad And Brain

Medaka is a model animal for vertebrate developmental biology, stem cells and germ cells research. Medakaisa small fish and have fast life cycle, inbred strains, mutant lines, complete genome sequence and numerous EST expression library. Unlike most fish, medaka with diploid chromosome structure, more interestingly, the Dmy gene located on the male Y chromosome determining the sex differentiation. Series of medaka cell lines, including the diploid embryonic stem cell lines, haploid stem cells, adult reproductive cells and neural stem cell lines from the eyes, etc. have been successfully established. These cell lines provides a good platform to study gene function by cytobiology. Medaka germ cells can labeled by specific gene promoter drived or specific 3 ’UTR combined GFP. Morpholino(MO) mediated gene knockout technology has been widely used in medaka. Recently, the rise of gene editing techniques, including ZFN and TALENs, especially the CRISP Cas9 technology, bring us effective apporch to detect the gene function and molecular mechanism involved in developmental processes. These techniques have been carried out in medaka. More and more domestic scholars that research reproduction and developmental of vertebrates use medaka as the model animal.Mature micro RNAs(mi RNAs) are a class of evolutionally conserved, singlestranded, small(approximately 19-23 nucleotides), endogenously expressed, and nonprotein-coding RNAs that act as post-transcriptional regulators of gene expression in a broad range of animals, plants, and viruses, in which involved ingerm cell proliferation, zygotogenesis and ontogeny processes.Gonadal development,where primary germ cells develop and differentiate, involves coordinated expression of thousands of genes. The spatiotemporaland dynamically expression of the related genes which is required for primary germ cell development, spermatogenesis, luteolysis, etc. are known to be under the control of posttranscriptional regulatory mechanisms. mi RNA as post-transcriptional regulators have been shown to be involved in this mechanism.Through Dicer, Drosha and DGCR8 knockout, key gene of mi RNAs synthesis pathways, lead to gonad function defects, stem cell differentiation abnormal, gametogenesis obstacles, eventually cause sterility phenotype. Proof the important biology role in the gonads of mi RNAs.In this study, we constructed four small RNA libraries from male and female O. latipes gonad and brain respectively for Solexa sequencing. A number of known and novel mi RNAs were identified and differently expressed anlysis was performed between the testis and ovarylibraries. Besides, we predicte thetarget of the differented expressed mi RNAsvia Targetscan and mi Randaand GO functional annotation and KEEG pathways analysis were performed. In addition, q PCR was carried out to confirm the sequce data and identifiy the tissue specific mi RNAs. It’s indicated that mir-202-5p restricted to gonad and expressed abundant in testis comparied to ovary and ISH were performed to detect the celluar location of mir-202-5p subsequently, meanwhile we adopt the use of antagomi Rs to knockdown levels of mi RNAs in medaka embryos.The main points are indicated as follows:A total of 22,764,505, 20,469,266, 14,691,690 and 10,305,258 raw reads were identified in the testis, ovary, male and female brain libraries, respectively. After data cleaning, a total of 13,481,015, 11,234,426, 13,092,735 and 9,802,297 clean reads were obtained from the four libraries. The majority of the small RNAs were 21~23 nt in size. Sequences 22 nt in length, the typical size of Dicer-derived products, accounted for over 35% of the total sequence reads in average of the four libraries. The results showed that mi RNAs were widely expressed in medaka gonads.After aligning the sequence data to mi RBase, 118 known mi RNAs corresponding to 77 pre-mi RNAs were identified, in which 11 were newly discovered. 696 new mi RNAs were predicted according the principle of mi RNAstructure. There are 29 significant difference expressed mi RNA between medaka testis and ovary: 16 were raised in testis and 13 down regulated. It is interesting that, the result of conservative analysis of significant differences mi RNAs reveal that medaka mi RNAs high conserved with salmon, followed by human, mice and show large variations compared with zebrafish.The GO and KEGG pathway analysis indicated that the target genes of the differencemi RNA bewteen testisand ovarylibrary were enriched in signaling pathways with reproductive processes including the Gn RH, Wnt, MAPK and TGF-beta.Using q PCR to detect 3 candidate mi RNAs(mir-202-5p, mir-219 c, mir-204) across 7 kinds of medaka tissues, the results indicated that the sequence data were receivable and mir-202-5p was gonad specific and mir-219 c was restricted to brain.Two color fluorescencein situ hybridization showed that mir-202-5p restricted to germ cells and expressed abundant in testis comparied to ovary. In vivo inhibiting medaka mir-202-5p lead to the decrease in the number of germ cells, migration anomalies, and unbalanced the male and female ratio. Reveals the mir-202-5p plays an important role in medaka sex differentiation, germ cell development, especially the testis development process.This study reveals the medaka mi RNAs high conserved with Salmo salar, and found 11 new mi RNAs, predict a large number of micrornas, enriched medaka mi RNA database, provide the basis for subsequent research on specific mi RNA functionof gonad in medaka. Simultaneously, there are multiple differences between male and female gonad, mir-202-5p was found to be involved in sex differentiation and germ cell development regulation, expand the knowledge of vertebrate sex differentiation.