Molecular Characterization, Expression And Functional Analysis Of Regulatory Region Of Germ Cell Marker Genes In Half-smooth Tongue Sole (Cynoglossus Semilaevis)

Primordial germ cells (PGCs) are the precursors of germ cells that migrate from theorigin site to the gonadal anlage and eventually give rise to gametes. Recently, thestudies of germ cell development and differentiation have become a new importantresearch area for the better understanding of sex differentiation mechanism.Investigations of the germ cell marker genes in fish will lay a solid foundation for thestudies on the mechanisms of germ cell differentiation, migration and development,and are essential for genetic resource conservation and fish reproduction. However,the research of germ cell markers is very limited in marine fish to date. Half-smoothtongue sole (Cynoglossus semilaevis) is one of the most valuable marine flatfish inChina. But females of the specie grow two to three times bigger and faster than males,low proportion of physiological female and limited the large-scale production of eggsunder artificial culture condition; these factors limit the rapid development of tonguesole culture. The all female production and surrogate brood technology might providean ideal solution to this problem, yet the lack knowledge of sex differentiation andlimited germ cell molecular marker have been reported in this species, which becomea key factor limiting the studies.In the present study, we cloned and characterized full-length cDNA and DNAsequence of vasa、nanos、dnd and pou2gene from tongue sole, studied the mRNAexpression levels in different tissues and embryonic stages by real-time quantitativePCR (qRT-PCR), and the sex-linked differential expression was also detected duringearly developmental and sex differentiation stages. The expression profile duringgametogenesis was investigated by in situ hybridization. In addition, we isolated theregulatory regions of vasa、nanos、dnd and pou2gene using Genome Walking andgenomic PCR, analyzed the potential transcript factor binding sites in5′flanking region by online software, and then constructed the plasmids containing regulatoryregions of vasa and nanos gene and green fluorescence protein (GFP) gene. Thefunction of regulatory regions was analyzed by microinjecting plasmid andsynthesized mRNA into fertilized medaka eggs. The study not only lay thegroundwork for PGCs labeling and manipulation (isolation, cryopreservation andtransplantation), but also provide basic data for investigating germ cell biology andcontrolling sex of half smooth tongue sole. The main results are summarized asfollows:1. The Vasa sequence of togue sole (CsVasa) contains highly conserved domains ofthe DEAD-box protein family, and CsVasa belonged to homologues of the teleostsand clustered with Senegalese sole (Solea senegalensis). The vasa gene (Csvasa) fromtongue sole was mainly expressed in gametes by in situ hybridization. qRT-PCRanalysis revealed that Csvasa expression was mainly restricted to the gonads,decreased during embryonic and early developmental stages, and increased with theprimordial germ cell proliferation. Csvasa mRNA expression levels in neomales weresignificantly lower than those in normal males and females. A typical sexuallydimorphic expression pattern of Csvasa was observed during early development andsex differentiation.2. The potential transcript factor (TF) binding sites were analyzed in5.2kb5′flanking region of Csvasa, and the pCsvasa-GFP-T and GFP-Csvasa3’UTR plasmidswere constructed. Medaka (Oryzias latipes) PGCs could be transiently labeled bymicroinjection of synthesized mRNA containing the green fluorescence protein geneand3’ untranslated region of Csvasa, and the Csvasa promoter could efficiently driveGFP expression in medaka embryos.3. The Nanos sequence of togue sole (CsNanos) contains two conserved CCHCzinc-finger domains, and CsNanos belonged to Nanos2cluster. The nanos gene(Csnanos) from tongue sole was mainly expressed in gametes by in situ hybridization.qRT-PCR analysis revealed that Csnanos expression was mainly restricted to thegonads, decreased during embryonic and early developmental stages, and increasedwith the primordial germ cell proliferation. Csnanos mRNA expression levels in neomales were significantly lower than those in normal males and females. A typicalsexually dimorphic expression pattern of Csnanos was observed during earlydevelopment and sex differentiation.4. The potential TF binding sites were analyzed in3.7kb5′flanking sequence ofCsnanos, and the GFP-Csvasa3’UTR plasmid was constructed. Medaka (Oryziaslatipes) PGCs could be transiently labeled by microinjection of synthesized mRNAcontaining the green fluorescence protein gene and3’ untranslated region of Csnanos.5. The Dnd sequence of togue sole (CsDnd) contains RNA recognition motif (RRM)and five conserved regions (NR, CR1~4), and CsDnd belonged to homologues of theteleosts. Two DNA fragments including2.7kb5′flanking region and1.8kb3′flanking region were obtained and the potential TF binding sites were analyzed in2.7kb5′flanking sequence of dnd gene (Csdnd) from tongue sole. The Csdnd wasmainly expressed in gametes by in situ hybridization. qRT-PCR analysis revealed thatCsdnd expression was mainly restricted to the gonads, decreased during embryonicand early developmental stages, and increased with the primordial germ cellproliferation. Csdnd mRNA expression levels in neomales were significantly lowerthan those in normal males and females. A typical sexually dimorphic expressionpattern of Csdnd was detected during early development and sex differentiation.6. The Pou2sequence of togue sole (CsPou2) contains highly conservedPOU-specific and POU homeo domains of the POU protein family, and CsPou2belonged to Pou2branch. The potential TF binding sites were analyzed in4.1kb5′flanking sequence of pou2gene (Cspou2) from tongue sole. qRT-PCR analysisrevealed that Cspou2expression was mainly restricted to ovary and no expression inmales and neomales, decreased during embryonic and early developmental stages, andincreased with the primordial germ cell proliferation. A typical sexually dimorphicexpression pattern of Cspou2was observed during early development and sexdifferentiation.