Study On The Developmental Mechanism Of Mouse Female Germline Stem Cells And Embryonic Stem Cells Based On Stemness Maintenance

Stem cells can self-renew and maintain the capacity of differentiation.Asymmetrical cell division plays critical regulatory roles for the maintenance of stem cell identity and multilineage differentiation.Thus,we study the developmental mechanism of stemness maintenance of adult stem cell(female germline stem cells)and pluripotent stem cell(embryonic stem cells).This will not only help us to understand the developmental process of mammalian stem cells,but also provide the theoretical basis for clinical application of stem cells.For years,the reproductive area always focuses on the regulation of stemness maintenance in germline stem cells.However,how the cells maintain stemness,the upstream/downstream signaling pathways and the interacted genes or proteins are well elucidated in spermatogonial stem cells,but not in female germline stem cells.To step forward to understand those unsolved questions,we report low-input cell transcriptome analysis of PGCs,FGSCs,GV and MII oocytes by performing RNA sequencing of freshly isolated cells in mice.This further demonstrates that FGSCs is a unique stage between PGCs and GV oocytes.Analysis of genome-wide DNA methylation during female germline development confirm the similarity of cultured FGSC and fresh FGSC,exhibiting low level of DNA methylation.By pathway analysis,we found PI3K-AKT gradually decreased during female germline development.Further,we confirmed this pathway is critical for FGSCs maintenance and proliferation by small molecule inhibition experiments.We also identify functional modules with hub genes that had the highest correlation with FGSC stage.Those genes represent potential novel candidates for regulating FGSC self-renewal and differentiation.Based on the dynamic expression of long noncoding RNAs(lnc RNAs),we identified 632 lnc RNAs,which were dramatically different and exhibited specific pattern for each developmental phase.Remarkably,we validated that Xist expressed in FGSCs and induced one X chromosome inactivation and we note alternative splicing patterns change dramatically in the female germline,with the highest occurring in FGSCs(57.12%),indicating more active gene regulation activity is required in FGSCs to maintain the stemness and differentiation potential.The regulation of stemness maintenance in embryonic stem cell is also a key question in stem cell biology.Although it is known that epigenetic mechanisms could affect the stem cell maintenance and lineage differentiation,we are still not clear about the transmission of epigenetic information.In recent years,emerging evidence points to an indispensable role for ACD and histone inheritance during stem cell development.Here we study the histone inheritance and the mitotic machinery with this model.We report here that DNA replication-dependent canonical H3/H4 show mutually exclusive regions for “old” vs “new” histones in asymmetrically dividing cells,either in chromosomal domains or in whole chromatids.However,H2 A,H2B and H3.3 show symmetric distribution.Remarkably,when wnt3 a beads are inactivated by DTT or m ESCs are cultured in complete LIF-2i medium,nonoverlapping histone pattern is lost.Because the proper organization of mitotic spindle is essential for normal separation and transmission of genetic information,we traced the centrosome,microtubule,nuclear membrane and centromeres in asymmetrically dividing m ESCs.The results showed that those components are all asymmetrically organized or distributed with Wnt3 a beads.We deduced that those asymmetric components of mitotic machinery could preferentially recognize and capture the chromosome with different histone inheritance,different genetic materials are thus distributed to different daughter cells.This work linked the histone incorporation with asymmetric cell division,and discovered a new mechanism to maintain the epigenetic memory and stem cell identity.In conclusion,we first studied the transcriptome and epigenome of early female germ cells with low-input cells RNA-Seq and Me DIP-Seq.We found PI3K-AKT pathway is critical for FGSCs maintenance,and thereafter we calrified the molecular,cell biological and epigenetic features of FGSCs.Moreover,we used asymmetrically dividing m ESCs and checked the histone inheritance and mitotic machinery,which is associated with stem cell maintenance.In this study,we used two different stem cells to decipher the key question “how stem cells maintain the stemness”,and found novel regulatory signaling pathway and epigenetic patterns.Together,this work serves as a new clue to study epigenetic mechanisms for maintaining the stem cell identity or differentiation in mammalian stem cells.