Study On The Regulation Mechanism Of C1EIP In Chicken PGCs Formation

Germ cell has an irreplaceable role in passage of the genetic information to the next generation and sustain life.Primordial germ cells?PGCs?are the ancestor cells of all germ cells can differentiate into spermatogonial stem cells?SSCs?or ovarian stem cells?OSCs?in the gonads,and further developed into sperm or ovum.The occurrence of PGCs is regulated by many exogenous factors such as endogenous regulatory factors and extracellular matrix.These factors form a complex regulatory network through different signaling pathways,directly or indirectly regulate the formation of PGCs.Currently,the researchers have done a lot of research on the growth and differentiation of PGCs by in-vitro isolation,in-vitro induction,construction of transgenic cells and so on.However,little is known about the molecular mechanism of regulation of PGCs,Therefore,the mechanism of PGCs has become the focus of attention in recent years.With the deepening of PGCs research,researchers have explored the discovery that there are key genes,signaling pathways,growth factors,and epigenetic factors that play important regulatory roles in this process.Although these factors have contributed to the occurrence of PGCs,there is no real sense to improve the efficiency of PGCs formation and thus can not meet the relevant research needs.Therefore,it is urgent to innovate the mechanism of PGCs from new fields to explore and understand the generation process of PGCs in order to obtain a large number of PGCs.Therefore,in order to further explore the mechanism of regulating the occurrence and differentiation of PGCs and fundamentally solved the problem of PGCs generation efficiency,based on RNA-Seq performed in chicken Embryonic stem cells?ESCs?,PGCs and SSCs transcriptome,we screened significantly different and PGCs' specific expressed gene,C1EIP?Genbank Accession:XM₄16629.3?in differentiation process of ESCs to SSCs and systematically explored its function and regulation mechanism in the process of differentiation of male germ cells from the two levels of in vivo and in vitro.The aim of this study is to understand the mechanism of differentiation of chicken ESCs into PGCs,provide a theoretical basis and reference for the effective differentiation of ESCs to PGCs and a theoretical basis for the study of the function and mechanism of other similar key functional genes for the differentiation of chicken ESCs into PGCs.The results are as follows:1.In order to effectively determine the biological function of the candidate specific gene C1EIP and its localization in eukaryotic cells and lay the foundation for the study of late function and mechanism,we constructed bioinformatics analysis and the results showed the C1EIP had a major high homology with birds,but also was unknown homologous proteins and was not found in specific domains of gene function and so require further study.we constructed the recombinant expression vectors pEGFP-C1EIP and pcDNA3.0-C1EIP by RT-PCR with amplification of C1EIP CDS squence provided by NCBI database.DF1 cells were transfected with pEGFP-C1EIP and pcDNA3.0-C1EIP to determine the localization of C1EIP and RT-PCR and Western Blot were used to detect the transcription and translation of C1EIP in eukaryotic cells.At the same time,the pcDNA3.0-C1EIP vector was encapsulated with PEI and injected into the mice intramuscular to prepare anti-mouse serum whose titer was detected by IFA and Western Blot.The results showed that the digestion and sequencing results indicated the pEGFP-C1EIP and pcDNA3.0-C1EIP vectors were successfully constructed.egfp expressed in the cytoplasm after transfection of DF1 cells with pEGFP-C1EIP,indicating that C1EIP is localized in eukaryotic cytoplasm and is capable of initiating transcription and translation.Anti-mouse serum was prepared by encapsulating pcDNA3.0-C1EIP product with PEI at a ratio of 1|?g:1.5?g.The IFA detection titer was 1:25 and the Westen Blot assay showed that the C1EIP protein was recognized.2.In order to explore the biological function of C1EIP in the process of chicken PGCs formation,we desigined 3 shRNA target sites and 1 negative control site for the C1EIP,and then.lentiviral interference vector was constructed and lentivirus was encapsulated.qRT-PCR was used to detect the interference efficiency of lentivirus interference vectors.In vitro,the 2nd generation ESCs were transfected with the lentiviral interference vector and overexpression vector respectively,and then induced by RA.ESC morphological changes were observed every 2 days,and the efficiency of PGCs formation was detected by qRT-PCR,cellular immunochemistry and flow cytometry.In vivo,the chick embryos were infected with the lentivirus interference vector,and the best infection efficiency was explored.Infected chick embryos were used to detect PGCs production efficiency by qRT-PCR,PAS staining,immunohistochemistry and flow cytometry analysis.The results showed that the lentiviral vectors C1EIP-sh1,C1EIP-sh2 and C1EIP-sh3 were successfully constructed and the interference efficiency was 80.39%,87.80%and 38.36%,respectively.In vitro,the normal RA induced the Embryoid Bodies?EBs?on the 2nd day.At the 4th day,the EBs became more and larger,and the edge of the EBs of the 6th day began to crack,and a small amount of cells were released.At the 8th day,the EBs were seriously ruptured and a large number of cells were released.At the 10th and 12th day,the EBs were completely cleaved and the SSCs-like cells appeared and grew together into grape strings.In overexpression group,large EBs appeared on the 2nd day,and small gaps appeared on the edge of the EBs at the 4th day.Most of the EBs on the 6th day were ruptured and released with a large number of internal cells.The SSCs-like cells were appeared on the 8th day and the typical grape string of SSCs clones were appeared on the 10th to 12th day.In knock-down group,the Ebs did not appear on the 2nd to 6th day.Until to the 8th day,the small EBs appeared.However,the number,size and morphology of the Ebs cultured to 12d did not show any significant changes.qRT-PCR results showed that overexpression of C1EIP resulted in the expression of sox2 was consistently decreased from 1.00±0.04 to 0.14 ± 0.01,and the expression of cvh,c-kit,stra8,Dazl,integrin a6 and integrin ?1 gradually increased to 2.14±0.03,2.79±0.05,2.26±0.06,2.73±0.03 and 2.59±0.05.The expression level of C1EIP was increased to 3.99 ± 0.06 on the 4th day and decreased to 2.87 ±0.07 at the 12th day.while the expression at the 8th to 12th day remained essentially unchanged,and the expression levels of c-kit,stra8,Dazl,integrin a6 and integrin ?1 were gradually increased to 2.14±0.03,2.79±0.05,2.26 ± 0.06,2.73 ± 0.03 and 2.59 ± 0.05.However,after knocking down C1EIP,the expression of sox2 did not change significantly until to the 12th day,and the expression of cvh,c-kit and C1EIP decreased from 1.00±0.04,1.00±0.04,1.00±0.03 and 1.00±0.04 to 0.53±0.04,0.45±0.05,0.86±0.03 and 0.76±0.02.stra8,Dazl,integrin ?6 and integrin ?1 expression firstly decreased from 1.00±0.04,1.00±0.04,1.00±0.03 and 1.00±0.04 to 0.53± 0.04,0.45 ± 0.05,0.86 ± 0.03 and 0.76 ± 0.02,and then slightly increased to 0.59±0.01,0.80 ± 0.04,0.88 ± 0.04 and 0.93 ± 0.08.The results of immunocytochemistry showed that the expression of CVH and C-KIT was significantly increased in the overexpression group compared with the RA-induced group,while the knockdown group inhibited the expression of CVH and C-KIT.Flow cytometry analysis results showed that the proportion of CVH+ cells in the RA-induced group was 3.4%,and the percentage of CVH+ cells was significantly up-regulated to 4.6%after overexpression of C1EIP,however,knockdown of C1EIP reduced CVH+cells to 2.9%.In vivo,the best conditions for chicken embryos to inject lentiviral vectors were blunt-ended 10 ?L 106 TU/mL lentiviral vector + 90 ?L DMEM.After injection,EGFP could be detected by PCR in chicken embryos and green fluorescence could be seen.The results of qRT-PCR showed that there was no significant change in the expression level of each gene between the blank group and the sh-Ctrl group.The decrease of the expression of sox2 in knock-down group were lower than that in other groups,and the expression of cvh,c-kit,Stra8,Dazl,integrin?6,integrin ?1 and C1EIP decreased significantly to 0.20 ± 0.07,0.43±0.08,0.45 ± 0.06,0.51±0.07,0.35 ± 0.07,0.04 and 0.22±0.03.PAS results show that knock down C1EIP can significantly reduce PGCs production.Immunohistochemical results showed that CVH and C-KIT were highly expressed in the reproductive ridge of the blank group and the negative control group,and the expression site was almost filled with the whole reproductive ridge.However,the expression of CVH and C-KIT was significantly decreased in the knock-down group,and the site of expression is confined to the edge of the reproductive ridge.The results of flow cytometry showed that the knockdown of C1EIP resulted in a significant reduction in cvh-labeled PGCs to 3.1%relative to the blank and control groups?4.6%and 4.3%?.3.In order to systematically elucidate the transcriptional regulation mechanism of C1EIP expression,we screened about 2000bp sequence upstream of C1EIP transcription start site provided in NCBI and UCSC database,the long fragment of C1EIP promoter was amplificated and cloned into pEGFP-N1 vector for replacing the CMV promoter to constructe recombinant expression vector pC1EIP-EGFP,which was transfected into DF1 cells to detecte the promoter activity of the long fragment of the C1EIP promoter.C1EIP promoter with different deletion fragment vectors PGL3-P1-PGL3-P10 were constructed with deletion fragment cloning technique.After transfection of DF1 cells,the promoter activity of each deletion fragment was detected by double luciferase reporter system,and the core regulatory region of C1EIP promoter was screened.In the core regulatory region,bioinformatics was used to predict the key transcription factor binding sites.The deletion vectors of the transcription factor binding sites were constructed and the effect of deletion of transcription factor binding sites on the activity of the promoter was also examined by double luciferase reporter system.Finally,The effects of methylation and acetylation on the promoter activity of C1EIP and C1EIP expression in ESCs,PGCs and SSCs were detected with lOpmol/L 5-Aza-2'-deoxycytidine?5-Azadc?,1?mol/L Trichostatin A?TSA?and 4mmol/L Valproic acid?VPA?treatment.The results of digestion and sequencing showed that pC1EIP-EGFP was constructed correctly and transfected into DF1 cells to stimulate the green fluorescence.It was qualitatively indicated that the extended fragment of the amplified C1EIP promoter had the activity of initiation.Simultaneously,the results of restriction enzyme digestion and sequencing of the deletion fragment of the promoter also indicated that the vector was constructed successfully.Double luciferase assay showed that-1025?-912bp was the core active region of the C1EIP promoter and the key transcription factor binding sites in this region include MEIS1,MAFG::NFE2L1,STAT3,HLTF and Hand1::Tcf3,in which only STAT3 plays a positive regulatory role.Finally,It was found that the promoter activity of C1EIP increased from 10.82±0.54 to 18.49 ± 0.72,33.27±0.83 and 38.11±0.53 after methylation inhibition of 5-Azadc and acetylation inhibitor TSA and VPA.The expression of C1EIP in chicken ESCs,PGCs and SSCs was changed from 1.00±0.23,5.73± 0.54 and 0.94 ±0.23 to 1.43 ± 0.42,10.60±0.26,2.83 ± 0.18 and 2.64 ± 0.23,9.89±0.37,3.25±0.16 and 2.99±0.43,8.33 ± 0.56,2.94 ± 0.29 after treatment with 5-Azadc,TSA and VPA,respectively.4.In order to explore the mechanism of C1EIP regulation of PGCs,we constructed C1EIP prokaryotic expression vector and induced it to express in E.Coli and digged out C1EIP interaction protein by GST pull-down.The fusion expression vector pcDNA3.0-ENO1-HA was constructed and used for in vitro Co-immunoprecipitation to verify the interaction between ENO-HA and C1EIP-EGFP.The interaction protein of ENO1 were explored by bioinformatics analysis,and then their expression changes in the process of PGCs formation was confirmed by qRT-PCR.Myc promoter double luciferase reporter vector PGL3-Myc-pro and recombinant expression vectors pcDNA3,0-MBP-1.At the same time,in order to eliminate the interference of homologous family genes,we also constructed recombinant expression vector pcDNA3.0-ENO2.pcDNA3.0-ENO1-HA,pcDNA3.0-MBP-1 and pcDNA3.0-ENO2 were co-transfected with PGL3-Myc-pro respectively and the activity of Myc promoter was detected by double luciferase reporter system.Simultaneously,based on the results obtained and the related literature,the pattern of PGCs generated by C1EIP and ENO1/MBP-1 mediated Notch signaling pathway was drawn.GST pull-down results showed that there was interaction between ClEIP and ENO1,and in vitro Co-IP was used to verify the interaction between C1EIP and ENO1.Through the Uniport and PSORT II database and the co-expression of network analysis,we found that ENO1 located in the cytoplasm and is associated with Myc,whose downstream gene is Notch1.The results of qRT-PCR showed that C1EIP and ENO1 were consistent in the differentiation of chicken ESCs into SSCs and were opposite to those of Myc and Notch1.The results of digestion and sequencing showed that the double luciferase reporter vector PGL3-Myc-pro and the recombinant expression vectors pcDNA3.0-MBP-1 and pcDNA3.0-ENO2 were all constructed successfully,and the double luciferase results showed that MBP-1 had an inhibitory effect on the activity of Myc promoter,while ENO1 and EN02 had no effect on it.Finally,according to the above results and related literature,we draw the pattern that C1EIP interactd with ENO1/MBP-1 to mediate Notch signaling to regulate PGCs generation.As an anchor protein,C1EIP immobilized ENO1 in the cytoplasm.When ENO1 is phosphorylated or dephosphorylated and translocated into the nucleus and lost the previous process of 96 amino acid residues to form MBP-1 protein,which combined to Myc promoter,which inhibited Myc transcription and blocks the signal transduction of Notch signaling pathway,eventually promoted the differentiation of chicken ESCs into PGCs.