Revealing cellular and molecular transitions in neonatal germ cell differentiation using Single-cell RNA sequencing

Neonatal germ cell development provides the foundation of spermatogenesis. However, a systematic understanding of this process is still limited. To resolve the cellular and molecular heterogeneity, we profiled single-cell transcriptomes of undifferentiated germ cells from neonatal mouse testes and employed unbiased clustering and pseudotime ordering analysis to assign cells to distinct cell states in the developmental continuum. We defined the unique transcriptional programs underlying the migratory capacity, resting cellular states and apoptosis regulation in transitional gonocytes. We also identified a subpopulation of primitive spermatogonia marked by CD87/uPAR, which exhibited a higher level of self-renewal gene expression and migration potential. We further revealed a differentiation-primed state within the undifferentiated compartment, in which elevated Oct4 expression correlates with lower expression of self-renewal pathway, higher Rarg expression, and enhanced retinoic acid responsiveness. Lastly, the knockdown experiment revealed the role of Oct4 in the regulation of gene expression related to the MAPK pathway and cell adhesion, which may contribute to stem cell differentiation. Our study thus provides novel insights into the cellular and molecular regulations during early germ cell development. Here, we performed single-cell RNA-Seq of germ cells from mouse testes on postnatal day (PND) 5.5. We also obtained transcriptomes of subpopulations marked by different levels of CD87 or Oct4-GFP, as well as SSC culture after Oct4 knockdown by bulk RNA-Seq.