Cell-Fate Transition And Determination Analysis Of Mouse Male Germ Cells Throughout Development

During mammalian male germ cell development,starting cells undergo stepwise cell-fate transitions to form functional sperm,but the full-term developmental program of male germ cells has not been catalogued.Here the transcriptome atlas of 11,598 individual cells covering 28 critical time-points throughout mouse germline development are interrogated,presenting an unbiased male germ cell developmental landscape.Although previous studies have provided much knowledge about germline development such as PGC specification and epigenetic reprogramming as well as PGC-to-gonocyte transition,it remains largely uncharacterized about the transcriptome atlas of full-term male germline cycle and how stepwise cell-fate transitions orchestrate male germ cell development.In the present study,we established the full-term single-cell transcriptome atlas and cell-fate transition landscape of mouse male germline cycle,and revealed several critical developmental stages/regulators for cell-fate determination.(1)To the best of our knowledge,our study is the first to resolve the whole developmental process of mouse male germ cells and provide so far the most comprehensive high-precision single-cell transcriptome reference map,which was composed of 11,598 single-cell high-quality transcriptomes(an average of 9,413 detected genes and 302,772 mRNA molecules detected per cell)and covered nearly all stages of male germ cells(from specification PGCs to round spermatids).The unbiased single-cell transcriptome atlas of male germline cycle established here allowed for an accurate overview of the cell-fate transition and determination underlying male germ cell development.For easier access to this resource,we have constructed a website at https://tanglab.shinyapps.io/Mouse_Male_Germ_Cells/.It should be convenient to know the dynamic expression pattern of any candidate gene for the guidance of gene knockout assay;especially,it can be used to dig out more lowly-expressed functional genes like transcription factors.In addition,our dataset should be a comprehensive reference for comparison to inform the accurate cell type-specific transcriptional changes when any interesting gene is manipulated in male mouse germline.It also paves avenues for integrating other molecular layer information to decipher the regulatory networks for cell-fate decision in germline development.(2)Based on the unbiased transcriptome reference map for the full-term developmental process of male germ cells,a global transcriptional reconfiguration was observed in the process of cell-fate transition from mitotic to mitotic arrest PGCs,wherein a transitional PGCs progression was uncovered to be crucial for this process.Notably,Hesl,a canonical downstream effector of Notch signaling pathway,was highly expressed in transitional PGCs,suggesting the potential functions of Hesl or Notch1-Hes1 signaling pathway in the transition from mitotic to mitotic arrest PGCs.Mechanistically,Notch signaling pathway was proved to exert essential roles in regulating the initiation of mitotic arrest,wherein inhibition of Notch signaling pathway leaded to the failed decrease of Mki67 positive PGCs,and eventually contributed to the formation of the spermatogonial stem cell pool.The role of cell cycle regulation in proper cell-fate transition was further demonstrated by Helq knockout mouse model;in detail,Helq ablation leaded to an aberrant progression of transitional PGCs and developmental delay into post-mitotic PGC stage,which eventually caused male subfertility.Of note,Helq deficiency also caused an abnormal transcriptional reprogramming of gene modules that was crucial for the mitotic to mitotic arrest transition.In summary,our studies based on Notch signalling pathway and Helq ablation demonstrated the critical role of proper cell-fate transition from mitotic to mitotic arrest PGCs in ensuring male fertility.Further research on the newly defined cell stages or dissecting potential regulators using our datasets should reveal the important nodes governing cell-fate transitions of germ cells.(3)The evolution of germ cell development is a matter of intense debate and the production of human haploid germ cells in vitro could hitherto not be accomplished.To shed lights on these fundamental questions,we used our high-resolution transcriptional reference map to carry out a careful comparison of mouse and human expression profile.We identified a set of conserved regulators that are likely the key drivers underlying cell-fate transition process.Interestingly,we screened out 435 male infertility and testicular germ cell tumor-related genes from the public database,which were also identified to be critical for the mitotic to mitotic arrest transition.The identification of these novel regulators could help to prioritise candidate factors that inform the forward induction of germ cell states in vitro to solve long-standing problems such as the generation of human haploid germ cells in a dish.Collectively,this study offers an accurate transcriptome atlas of male germline cycle and allows for an overview of the cell-fate transition and determination underlying male germ cell development.