The Role Of Diaph1 In Migratiory Mouse Primordial Germ Cells

Primordial germ cells(PGCs)are the precursor cells of sperm and ovum.The migration of PGCs from the specialization sites to the genital ridge is finely regulated,which is determined by its own cell characteristics and the cross-talking with surrounding environment and accompanied by cell proliferation and apoptosis.At present,the molecular mechanism of PGCs migration remains to be further studied.In this study,the differentially expressed genes were obtained by analyzed the RNA-seq data of mPGCs at different developmental stages,and candidate genes were selected after KEGG pathway analysis.The expression and function of Diaph1 gene were analyzed with Diaph1 knockout mouse strains,the effect of Diaph1 on mouse fertility and growth,as well as the on number,proliferation,apoptosis and adhesion of mPGCs were studied.In this study,the location of mPGCs and the selection markers were studied by detecting the expression of different molecules.By using immunofluorescence method with tissue secyion,the results showed that Oct4 can be used as a marker for mPGCs localization and sorting.The flow cytometry were used to sort mPGCs from the embryonic genital ridge of the Oct4-labeled mouse,the sorting efficiency was about 0.20% for E9.5 embryos,and about 2.28% for E12.5 embryos.When Ssea1 antibody was used as marker,the sorting efficiency of mPGCs in E12.5 were2.91%.After sorting,the cells were positive blue-purple after Alkaline Phosphatase staining.qPCR results showed that mRNA expression of the following gene were was significantly higher in positive cells than in negative cells,including,mPGCs marker genes Fkbp6,Mov101,Spo11 and 4930432K21 Riken,pluripotency genes Nanog and Oct4,reproductive-specific genes Mvh and Dazl,etc.Therefore,both Oct4 and Ssea1 can be used as markers for identification and sorting of mPGCs.This study analyzed our previous RNA-seq data of mPGCs at the E9.5 and E12.5,the differentially expressed genes were enriched into regulatory pathways.The genes of key nodes in ErbB signaling pathway and Focal adhesion pathway were selected for qPCR verification,and the expression change trend was consistent with the sequencing results.Subsequently,the literature analysis was performed to screen out the candidate genes,Diaphanous-related formins 1(Diaph1)and yippee like 1(Ypel1),associated with cell migration.The mRNAs expression of both gene wer increased in migrating mPGCs,while decreased after reached genital ridge,and had significant differences.To further verify the role of Diaph1 and Ypel1,knockout mouse were by CRISPR/Cas9 technology,respectively,and the effect on individual,tissue and mPGCs characteristics and the mechanisms were studied.The results showed that the fertility were reduced after knockout,especially in Ypel1 knockout mouse,could not support the further studies.The results from the reproduction experiments found that the genotypes of Diaph1 knockout mouse did not comply with Mendel's genetic law,indicating that knockout had an impact on fertility.Compared to be wild-type mouse,in knockout mouse,the weight of testis and ovaries decreased significantly,the number of mPGCs decreased,and the expression of Dicer1 and Mcm9 genes which related to proliferation was down-regulated.TUNEL experiments showed that the number of mPGCs,which undergone apoptosis were increased in the knockout mouse,and the expression of Map2k5 and Rest genes which maintained cell survival were down-regulated.Theexpression of E-cad protein and Cdh1 which related to adhesion were down-regulated,and the expression of Cxcr4,Hmgcr and Dazl genes which related to migration were down-regulated.In conclusion,Diaph1 is association with mPGCs migration,mRNA expression is higher in migrating mPGCs,and down-regulated after migration.After knocking of Diaph1,the reproductive capacity of mouse declined,the proliferation of mPGCs was inhibited,the apoptotic cells increased and the number of mPGCs decreased,in addition,the expression of genes releated to cell adhesion and migration-related were inhibited.This study laid the foundation for further understanding the molecular mechanism of mPGC migration.