Expression Regulation Of Dnd From Nile Tilapia And Its Role In Germline Cell Induction

In the early stage of embryonic development,germ cell lines are separated from somatic cell lines,which plays an important role in promoting the formation of the offspring by producing gametes.The ancestor of germ cell is primordial germ cell(PGC),which suffer several stages including specialization,migration and proliferation in the early stage of embryonic development.Following with the development of gonad,PGC can differentiate into differential germ cells at different stages,and finally format the sperm and egg respectively.Dead end(dnd)was firstly identified as a specific maternal RNA in the germ plasm of zebrafish,and it's a kind of RNA binding protein specifically expressed in germ line cells.Subsequently,it was found in other vertebrates.Dnd protein is very important for the migration and activity of PGC,and also plays an important role in the development of germ cells.In zebrafish,knockdown of dnd didn't affect the specialization and formation of PGC,whereas the PGC could not migrate to gonadal primordium,and finally it was apoptotic.At the early stage of medaka embryo development,dnd was mainly expressed in a few cells,while it was specifically expressed in germ cells.In medaka,knockdown of dnd could prevents the formation of PGC precursor cells while overexpression result in an increase of the number of PGC.On the other hand,many studies revealed that overexpression of dazl,vasa and another germ cell-specific genes in embryonic stem cells could induce the formation of germ cell-like cells.The Nile tilapia(Oreochromis niloticus),belonged to the Perciformes Cichlidae,is well known for its fast growth,strong resistance to stress and short-term reproductive cycle(14 days).It is an economically important fish in aquaculture all over the world.Our previous studies showed that tilapia dnd was specifically expressed in adult gonads,and dnd knockout tilapia using CRISPR/Cas9 technology leaded to the disappearance of germ cells in chimerism F0,suggested that dnd play an important role in the development of germ cells.However,the dnd expression pattern in the early stage of embryonic development,its regulatory mechanism are still unclear.At the same time,its expressions in response to Pou5f3,Sox2,retinoic acid(retinoic acid,RA),and estrogen are also still unknown.Hence,the biological roles of the tilapia dnd in the differentiation and development of PGC need to be further studied.In present study,tilapia was used as a reasearch model to study the expression and regulation of dnd using situ hybridization,immunohistochemistry and promoter analysis technologies.In addition,dnd overexpression,RT-PCR were used to investigate its biological roles in the differentiation and development of germ cell.The results are shown as follows:1.Expression patterns of dnd in tilapiaThe expression of dnd in tilapia embryo was detected by RT-PCR at different stages of embryonic development.Results showed that dnd was expressed only in testis and ovary in adult tissues while no expression was detected in other tissues.Additionaly,dnd was highly expressed in the early stage of embryonic development(1-24 h after fertilization),and then it continuouslly expressed until the gastrula stage,but no expression was detected after 24 h.Furthermore,immunohistochemistry showed that Dnd protein was expressed in all cells at blastocyst stage,which was consistent with the findings in zebrafish.In situ hybridization showed that dnd mRNA was only expressed in germ cells but not somatic cells in gonad tissue of adult tilapia.2.The transcriptional regulation of dnd2.1 Construction and transcriptional activity of pT2AL-Ondnd1.2-GFPA DNA sequence of 1.2 kb long from-1 to-1200 upstream of tilapia dnd next to the start codon(ATG)was successfully identified and cloned from tilapia genome,and its transgene reporter vector pT2AL-Ondnd1.2-GFP was constructed.The expression of endogenous dnd was detected by RT-PCR.The results showed that it was significantly expressed in tilapia embryonic stem cell line(TES1)and medaka spermatogonia cell line(SG3),while no expression was found in tilapia stromal stem cell line(TSL)and Southern catfish ovarian granulosa cell line(SCO).48 h after transfection with pT2AL-Ondnd1.2-GFP,obvious green fluorescence was observed in all the four types of cells,indicated that it could not regulate the expression of endogenous dnd in the cells.2.2 Construction and transcriptional activity of pT2AL-Ondnd1.2-GFP-3'UTRPrevious studies have shown that 3'UTR of germ cell-specific genes such as vasa and nanos enable specifically expressed in germ cells.In addition,Dazl 3'UTR was found has a function of stabilizing Dazl expression in mice.Thus,we isolated 1500 bp of the 3' UTR sequence of dnd and then cloned it into the downstream of the GFP stop codon of pT2AL-Ondnd1.2-GFP,named as pT2AL-Ondnd1.2-GFP-3'UTR.Then it was transfected into TES1,TSL and SG3,results showed that green fluorescence was significantly enhanced compared to pT2AL-Ondnd1.2-GFP.Thus,it suggested that the 3'UTR of dnd could significantly enhance mRNA stability,but it could not be specifically expressed in germ cells.2.3 Construction and Transcriptional activity of pT2AL-Ondnd1.3-GFPThe DNA sequence of the tilapia dnd 5' flanking from-1200 to+100 with a length of 1.3 kb was isolated and cloned,and then its transgenic reporter vector pT2AL-Ondnd1.3-GFP was constructed,and subsequently the vector was transfected into TES1,TSL,SG3 respectively.After 48 h of transfection,no expression of fluorescent protein was observed.The mRNA level of pT2AL-Ondnd1.3-GFP was detected by RT-PCR after 48 h of transfection.Results showed that the mRNA level was similar to its level in pT2ALOndnd1.2-GFP,and the sequencing results further verified the correctness of the sequence,suggested that the DNA sequence from +1 to +100 at the 5' flanking of dnd was involved in the post-transcriptional regulation of dnd.2.4 Construction and transcriptional activity of pT2AL-Ondnd1.3-GFP-3'UTRThe 1500 bp long 3' UTR sequence of dnd was cloned into the downstream of the GFP stop codon of pT2AL-Ondnd1.3-GFP,and it was named as pT2AL-Ondnd1.3-GFP-3'UTR.Then the vector was transfected into TES1,TSL and SG3.Results showed that fluorescence was observed in TES1 and SG3,while it was not detectable in TSL.These findings demonstrated that the 3'UTR and +1 to +100 DNA sequences jointly regulate the specific expression of dnd in germ cells.3.The dnd promoter activity analysis3.1 Luciferase activity analysis of pdnd-1.2luc and pdnd-1.8lucThe DNA sequence of 1.2 kb and 1.8 kb upstream of tilapia dnd next to ATG codon were isolated and cloned,and their luciferase reporter vectors were constructed,named pOndnd-1200 and pOndnd-1884 luc respectively,Then,TES1 cells were transfected,and the activity of the promoter region was quantitatively detected by measuring the fluorescence intensity.Results showed that pOndnd-1200 luc and pOndnd-1884 luc had stronger luciferase activity than the control group(p < 0.05),and the activity of p Ondnd-1884 luc was significantly higher than that of p Ondnd-1200 luc.The results showed that there was a positive regulatory sequence between-1200 and-1800.3.2 The regulation role of RAAfter transfection of TES1 with pdnd-1.8luc,it was treated with 100 nM,1 ?M,and 5 ?M RA(retinic acid,which promoted cell differentiation)for 3 days.Results showed that luciferase activity of the cells induced by RA was lower than that of the control group(p < 0.05),suggested that RA could negatively regulate the dnd expression.3.3 The negative regulation of Pou5f3 and Sox2After cotransfected TES1 with pdnd-1.8luc vector and eukaryotic expression vectors pcDNA3.1-pou5f3 and pcDNA3.1-sox2,Pou5f3 and Sox2 could significantly reduce luciferase activity(p < 0.05),results showed indicated Pou5f3,Sox2 could negatively regulate the dnd expression.3.4 The negative regulation of estrogenAfter cotransfected TES1 with pdnd-1.8luc vector and eukaryotic expression vectors pBIND-ER?-LBD?pBIND-ER?1-LBD and pBIND-ER?2-LBD,Results showed that compared with the control group,E2 could significantly reduce luciferase activity(p < 0.05),suggested E2 could also negatively regulate the dnd expression.4.Dnd was overexpressed in TES1 in vitro4.1 Construction of pCMV-Ondnd-DsredThe sequence of dnd was cloned from tilapia gonad tissue and successfully constructed into eukaryotic expression vector driven by CMV,and then the vector was named as pCMV-Ondnd-Dsred.4.2 Expression of pCMV-Ondnd-Dsred in TES1The reconstructed vector was transfected into TES1,and then the cells were cultured for 7days.Results showed that red fluorescence was observed in most(over 50%)cells.4.3 Expression of germline cell related genes in transfected cells.After 7 days of pCMV-Ondnd-Dsred transfection,the expression of early germ cell marker gene blimp1 was detected by RT-PCR,suggesting that Dnd may play a role in the induction of germ cells.