Preliminary Studies On Specification And Development Of The Primordial Germ Cells From Larimichthys Crocea By Nanos And Dnd Gene

In many fishes,germ cells are originated from primordial germ cells(PGCs)and set aside from somatic cell lineage in early embryonic development.The PGCs go through the specification and migration stages and eventually reach their targets where the gonad develops,i.e.,gonad anlag or primordial genital ridge.And then germ cells enter into the first stages of gametogenesis,namely the spermatogonia and oogonia.Through the processes of gametogenesis,they are maturing finally into spermatozoa and ova.As we know,germ cells have specific gene expression patterns,and some genes expressed specifically in germ cell,such as vasa,dead end(dnd),nanos3,dazl,and piwi.Mounting researchers use germ cell-specific expressing genes as a marker to study the specification and development of germ cell,since the vasa gene has been successfully applied in the study of PGCs of zebrafish(Danio rerio).Nanos gene was first demonstrated to participate in the establishment of the anterior-posterior body axis of Drosophila melanogaster embryos by inhibiting translation of hunchback mRNA.Subsequent researches demonstrated that it also takes part in the migration and maintenance of PGCs and is required for the self-renewing of germ stem cells.Dnd(dead end),a maternal gene that exclusively expressed in PGCs and encodes an evolutionary conserved RNA-binding protein,plays a critical role in germline cell development during embryogenesis.In this study,three nanos gene subtypes and dnd gene namely Lcnanos1,Lcnanos2,Lcnanos3,and Lcdnd from Larimichthys crocea were cloned by means of SMART-RACE.Meanwhile,the expression level of Lcnanos and Lcdnd were analyzed by qRT-PCR(real-time quantitative PCR)and WISH(whole mount in situ hybridization).Based on the expression patterns of Lcnanos3 and Lcdnd during embryogenesis,the origin and migration of PGCs were analyzed.The results were as follows:1)The full-length cDNA of Lcnanos1 was 1296 base pairs(bp)encoding 225 amino acid residues and was deposited in GenBank database under accession number KF690631.1.The cDNA sequence of Lcnanos2 was deposited in GenBank database under accession number KJ144823 and was 948 bp encoding 175 amino acid residues.The full-length cDNA of Lcnanos3 was 1519 bp encoding 223 amino acid residues and was deposited in GenBank database under accession number KF920597.1.Results showed that the deduced amino acid sequences of three Lcnanos have two consecutive conserved Cys-Cys-His-Cys zinc finger motifs.Besides,the full-length cDNA sequence of Lcdnd was 1359 bp encoding 380 amino acid residues.Moreover,the deduced amino acid sequences of Lcdnd have two RNA recognition motifs(RRMs)or RNA binding domains(RBDs)which were conserved among species and were required for the maintenance of germ cells of vertebrates.2)The expressions of mRNAs in different tissues were measured by qRT-PCR.Results showed that expression levels of Lcnanos1 in testis was significantly higher than other tissues(P<0.05),followed by heart,brain,Eye,and ovary.Nevertheless,both Lcnanos2 and Lcnanos3 were specifically expressed in gonad and they highly expressed in testis and ovary,respectively(P<0.05).The differential expression patterns among the three Lcnanos implied their different functions.Similar to Lcnanos3,Lcdnd specifically expressed in gonad and expression level in ovary was significantly higher than in testis(P<0.05).3)The spatio-temporal expression patterns of Lcnanos and Lcdnd were analyzed by WISH during embryogenesis.Results showed that there were no signals of Lcnanos1 and Lcnanos2 were detected at all developmental stages during embryogenesis(data not shown in this study).On the contrary,the signals of Lcnanos3 and Lcdnd were detected in all stages examined and their expression patterns were the same.Meanwhile,their expression patterns will contribute to elucidate the specification and development of PGCs.Results of WISH showed that both Lcnanos3 and Lcdnd mRNAs were localized to the cleavage furrow during early cleavage stages.As the embryo developed to blastula stage and early-gastrula stage,the increasing positive signals started to locate in a small number of cells(cell size: about 10 μm)of germ ring that means the specification of future primordial germ cells.Moreover,the positive cells were mainly distributed in the mesentoderm of germ ring region at early-gastrula stage.During subsequent stages,putative increasing PGCs migrated to the embryonic shield region along the germ ring and then had increased at both sides of the trunk.At somitogenesis stage,the positive cells were distributed as two lines located in lateral mesoderm and cell diameter were 10-11 μm.Subsequently,those cells migrated toward the abdomen of the trunk along the dorsal-ventral axis and then clustered in the putative primitive genital ridge.In short,herein,Lcnanos3 and Lcdnd were identified as maternal germ plasm components which specifically expressed in germ cell.Based on the expression patterns of Lcnanos3 and Lcdnd during embryogenesis,the specification of PGCs of L.crocea occurred at blastula stage and the specification pattern belong to preformation.Furthermore,we preliminarily analyzed the migration route of PGCs in embryos of L.crocea.This study laid the foundation for studies on specification and development of germ cell and the fertility control.