Dissecting gonadal cell lineage specification and sex determination during human development using a single-cell transcriptomics approach

Gonadal somatic cells are the main players in gonad development and are important for sex determination and germ cell development. Here, using a time-series scRNA-seq strategy, we analyzed the fetal germ cells (FGCs) and gonadal somatic cells in human embryos and fetuses. Clustering analysis of testes and ovaries revealed several novel cell subsets, including POU5F1+SPARC+ FGCs and KRT19+ somatic cells. Furthermore, our data indicated that DLK1+ cells may be the progenitors of steroidogenic cell lineages in both sexes and that TAC1+ cells may be the progenitors of granulosa cells in females. Intriguingly, the testosterone synthesis function transitioned from fetal Sertoli cells to adult Leydig cells in a step-wise manner. Moreover, interactions between gonadal somatic cells were systematically explored and verified in our study. In detail, we observed that Sertoli cells interacted with Leydig cells through DHH-PTCH1 and PDGFA-PDGFRA/PDGFRB ligand-receptor gene pairs. More importantly, we identified cell type-specific developmental defects of both FGCs and gonadal somatic cells in a Turner syndrome embryo (45, XO). Our work provides a blueprint of the complex yet highly ordered development and interactions of human FGCs and gonadal microenvironment cells. To further expand our understanding of the specification of gonadal somatic cells and sex differentiation in humans, we performed large-scale single-cell RNA sequencing (scRNA-seq) of gonadal cells in both sexes in a time series spanning gonad development from 6W to as late as 23W of gestation. In total, we obtained 56,399 gonadal cells from 16 embryos. **The raw files have been uploaded to the Genome Sequence Archive (GSA) database (accession number HRA000344).