NANOS2 suppresses the cell cycle by repressing mTORC1 activators in embryonic male germ cells

•Single-cell RNA-seq data recapitulated sexual differentiation processes•Cell cycle state of developing germ cells was revealed by single-cell data•NANOS2 suppresses mTORC1 activators to induce cell arrest During murine development, male enter the mitotically arrested G0 stage, which is an initial step sexually dimorphic differentiation. The male-specific RNA-binding protein NANOS2 has a key role in suppressing cells. However, detailed mechanism how regulates remains unclear. Using RNA sequencing (scRNA-seq), we extracted each wild-type and Nanos2-KO testes that Nanos2 expression starts mitotic induces arrest. We identified Rheb, regulator mTORC1, Ptma as possible targets NANOS2. propose repression primary function it mediated via suppression activity through Rheb post-transcriptional manner. differentiated gametes sperm eggs originally differentiate from common precursors, primordial (PGCs). 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To address above question, first conducted double KO found not sole repression. explore more regulation, (scRNA-seq) functions previous study, demonstrated 3′UTR-dependent manner forced deleting 3′-UTR led failure expression, resumption mitosis, similar phenotypes presence hypothesized issue, generated knockout (dKO) E12.5 injecting tamoxifen (see STAR Methods). If main driver arrest, dKO should rescue phenotype. stained marker Ki67 active mitosis. Contrary our expectation, detected still positive (Figure 1A), suggesting sufficient promote Importantly, report excess increased responsiveness exhibited dispensable point. addition, signals 1B). quantification, proportion This suggests there than Dazl. investigate re-examined states Nanos2+/− E15.5. progression examined single spreading co-staining anti-phosphorylated histone H3 (pH3), cells, anti-REC8, meiosis, antibodies. testis, observed exhibiting (1) negative REC8 pH3, punctate pH3 1C). former type likely includes (negative Figure 1C), latter (mitotic 1C); antibody used exhibits G2 (Hayashi-Takanaka 2009Hayashi-Takanaka Yamagata Nozaki Kimura Visualizing modifications living cells: spatiotemporal dynamics phosphorylation interphase.J. Cell 2009; 187: 781-790Crossref (82) majority classified (97.1%), (97.7%), (97.6%) initiated E13.5, next NANOS2−/− addition consisted (3) REC8-single-positive (4) pH3-double-positive (meiotic never normal (Figures 1C S1). abnormal or dying (abnormal Based quantification analysis, difference NANOS2+/− already evident E13.5; 24.2% mitotic, although 75.8% REC8/pH3-double-negative (negative) raised possibility initiation. did greatly increase (79.1%). decreased 56.5% accompanied reduction probably due apoptotic death. method useful examine characteristics cell, accurate classify populations loss nuclear components M phase. further precisely, antibodies against GCNA, Ki67, analyzed their intensities fluorescence-activated sorting (FACS) E14.5. Owing variation developmental timing, results varied stages samples: approximately 20% based markers GCNA number NANOS2-positive gradually S2 S3). Of note, Ki67-positive begins prior downregulation 1D). 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