Figures S1CS16:Click here to view.(4.3M, pdf) Data S1. cycle is a primary function of NANOS2 and that it is mediated MD2-IN-1 via the suppression of mTORC1 activity through the repression of in a post-transcriptional manner. ((which is strongly expressed in germ MD2-IN-1 cells just after colonizing the gonads in both males and females (Seligman and Page 1998). DAZL was reported to act as a licensing factor for germ cells to initiate sexual differentiation (Gill et?al., 2011). It is required for germ cells to acquire competence to respond to RA to enter meiotic prophase in ovaries, whereas its expression is repressed by NANOS2 once germ cells enter the testes. As mitotic activity is resumed and STRA8 is upregulated in the absence of NANOS2, germ cells were thought to be feminized (Suzuki and Saga 2008). However, it is unclear whether the repression of DAZL by NANOS2 is responsible for the maintenance of mitotic arrest and repression of feminization. To address the above question, we first conducted double KO of NANOS2 and DAZL, and found that DAZL is not the sole factor responsible for cell cycle repression. To explore more factors responsible for cell cycle regulation, we conducted single-cell RNA sequencing (scRNA-seq) analyses and revealed that NANOS2 functions not only to maintain the MD2-IN-1 arrested cell cycle but also to induce mitotic arrest. Results suppression by NANOS2 is not sufficient to repress mitotic resumption In the previous study, we demonstrated that DAZL expression is suppressed by NANOS2 in a 3UTR-dependent manner (Kato et?al., 2016). The forced expression of DAZL by deleting its 3-UTR led to the failure of male-type gene expression, meiosis initiation and resumption of mitosis, similar phenotypes of germ cells even in the presence of NANOS2 (Kato et?al., 2016). Therefore, suppression by NANOS2 is hypothesized to regulate cell differentiation and the cell cycle in male germ cells. To address this issue, we generated and double knockout (dKO) germ cells only after E12.5 by injecting tamoxifen at E12.5 (see STAR Methods). If suppression by NANOS2 is the main driver of male-type gene expression and cell cycle arrest, the dKO should rescue the phenotype. We stained DNMT3L as a marker of male differentiation, STRA8 MD2-IN-1 as a marker of meiosis initiation and Ki67 as a marker of active mitosis. Contrary to our expectation, DNMT3L expression was not detected and STRA8 was still positive in dKO germ cells (Figure?1A), suggesting that the suppression of by NANOS2 is not sufficient to promote male germ cell differentiation. Importantly, a previous report demonstrated that excess DAZL expression increased the responsiveness of germ cells to RA (Kato et?al., 2016). However, the dKO cells exhibited STRA8 expression, suggesting that DAZL is dispensable for germ cells to respond to RA at this point. In addition, dKO cells exhibited positive signals for Ki67 (Figure?1B). After quantification, the proportion of mitotically MD2-IN-1 active cells in the dKO was similar to that in and testes from E13.5 to E15.5. The progression of mitosis and meiosis was examined in each single germ cell by spreading germ cells and co-staining with anti-phosphorylated histone H3 (pH3), a marker of mitotic cells, and anti-REC8, a marker of meiosis, antibodies. In in C1, and in C3, in C6, in C7, genes in C8, genes in both C3 and C9, genes in C10 and in C11, were listed. In addition, many genes exhibiting certain cell population-specific expression were observed, demonstrating our scRNA-seq data to be useful to analyze the cell population in developing Rabbit polyclonal to c-Kit gonads. Using known distinct genes as markers, we identified the germ cell population and other somatic cell populations (Figure?2B). Importantly, we were able to observe the heterogeneity of gene expression within each cluster, especially in C1, suggesting that more careful analysis can separate more detailed cell types. To evaluate whether our data can be useful to analyze detailed differentiation pathways, such as sexual differentiation, we extracted 4 major cell types in.