Analysis Of Gene Expression And Pathways Of Mouse Primordial Germs Cell In Migration

Primordial germ cells(PGCs)are progenitor cells of germline.In mouse,PGCs originate from the epiblast cells of early postimplantation,which responsed to the instructive signals from the extraembryonic tissues.After specialized,mPGCs(mouse PGCs)migrate to the genital ridge and participate in gonadal development.Until now the migration process of PGCs is complicated regulated,which is remained to be elucidated.In this study,PGCs were isolated from OCT-4 labeled mouse embryos in 9.5 days(E9.5)and in 12.5 days(E12.5)by fluorescence-activated cell sorting.The two groups represent the migrating phase and the post-migration stage,respectively.In order to exclude the affect of gender differentiation,E12.5 embryos were seperated into male and female groups.Subsequently,the mPGCs were subjected to single-cell RNA-seq analysis by using the hiseq4000 sequencing platform in combination with the PE101 sequencing method.A total of 13,977 transcripts were obtained from all samples,of which 9,822 were transcripts of known coding RNAs and 1,048 were unknown transcripts.Compared the data from E9.5 and E12.5 female embryos,7395 DEGs(differentially expressed gene DEGs)were identified,with 4792 DEGs up-regulated and 2603 DEGs down-regulated(FDR?0.001 and fold-change? 2).Compared the data from E9.5 and E12.5 male embryos,there are 7,289 DEGs,with 4,287 up-regulated and 3,002 down-regulated.To validate the results,26 mRNAs of DEGs were randomly selected to analize by real-time PCR.The results showed the same dynamic changes in gene transcription,indicating that RNA-seq results are reliable.In order to elucidate the regulatory networks during the migration of mPGCs,DEGs obtained from sequencing were annotated in 75 Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways by pathway analysis(P<0.01).In female,56 signaling pathways are involved in the migration of mPGCs;in males,56 pathways are involved.37 of these signaling pathways are shared by both female and female mPGCs during migration.These pathways involve 15 cell survival,14 cell proliferation,7 cell adhesions,5 cell migrations and 3 cell differentiations.The ErbBs signaling pathway is located in all the above types of signaling pathways.The analysis of the ErbBs signaling pathway and its upstream and downstream regulatory networks revealed that the Ras signal pathway,EGFR tyrosine kinase inhibitors block signal pathway and Focal adhesion signaling pathway are also changed at the same time.Then LRRC58 and RHOA were verified by immunofluorescence analysis and 8 genes were verified by real-time PCR,which were performed on the involved in above pathways.The verification results were consistent with the analysis results.Therefore,ErbBs may play a role in the migration of mPGCs.In this study,though the analyzed of gene expression profiles of mPGCs during migration,the results would laid a foundation for further study of the migration mechanism of mPGCs and elucidation of mouse gametogenesis and differentiation.