Gonadal sex determination represents a unique model for studying cell fate decisions. However, a complete understanding of the different cell lineages forming the developing testis and ovary remains elusive. Here, we investigated the origin, specification, and subsequent sex-specific differentiation of a previously uncharacterized population of supporting-like cells (SLCs) in the developing mouse gonads. The SLC lineage is closely related to the coelomic epithelium and specified as early as E10.5, making it the first somatic lineage to be specified in the bipotential gonad. SLC progenitors are localized within the genital ridge at the interface with the mesonephros and initially coexpressWnt4andSox9. SLCs become sexually dimorphic around E12.5, progressively acquire a more Sertoli- or pregranulosa-like identity and contribute to the formation of the rete testis and rete ovarii. Last, we found that WNT4 is a crucial regulator of the SLC lineage and is required for normal development of the rete testis.

Despite the importance of germ cell (GC) differentiation for sexual reproduction, the gene networks underlying their fate remain unclear. Here, we comprehensively characterize the gene expression dynamics during sex determination based on single‐cell RNA sequencing of 14 914 XX and XY mouse GCs between embryonic days (E) 9.0 and 16.5. We found that XX and XY GCs diverge transcriptionally as early as E11.5 with upregulation of genes downstream of the bone morphogenic protein (BMP) and nodal/Activin pathways in XY and XX GCs, respectively. We also identified a sex‐specific upregulation of genes associated with negative regulation of mRNA processing and an increase in intron retention consistent with a reduction in mRNA splicing in XY testicular GCs by E13.5. Using computational gene regulation network inference analysis, we identified sex‐specific, sequential waves of putative key regulator genes during GC differentiation and revealed that the meiotic genes are regulated by positive and negative master modules acting in an antagonistic fashion. Finally, we found that rare adrenal GCs enter meiosis similarly to ovarian GCs but display altered expression of master genes controlling the female and male genetic programs, indicating that the somatic environment is important for GC function. Our data are available on a web platform and provide a molecular roadmap of GC sex determination at single‐cell resolution, which will serve as a valuable resource for future studies of gonad development, function, and disease.

Sex determination in mammals depends on the differentiation of the supporting lineage of the gonads into Sertoli or pregranulosa cells that govern testis and ovary development, respectively. Although the Y-linked testis-determining gene Sry has been identified, the ovarian-determining factor remains unknown. In this study, we identified −KTS, a major, alternatively spliced isoform of the Wilms tumor suppressor WT1, as a key determinant of female sex determination. Loss of − KTS variants blocked gonadal differentiation in mice, whereas increased expression, as found in Frasier syndrome, induced precocious differentiation of ovaries independently of their genetic sex. In XY embryos, this antagonized Sry expression, resulting in male-to-female sex reversal. Our results identify −KTS as an ovarian-determining factor and demonstrate that its time of activation is critical in gonadal sex differentiation.

Summary: Despite the importance of germ cell (GC) differentiation for sexual reproduction, the gene networks underlying their fate remain unclear. Here, we comprehensively characterize the gene expression dynamics during sex determination based on single-cell RNA sequencing of 14,914 XX and XY mouse GCs between embryonic days (E) 9.0 and 16.5. We found that XX and XY GCs diverge transcriptionally as early as E11.5 with upregulation of genes downstream of the Bone morphogenic protein (BMP) and Nodal/Activin pathways in XY and XX GCs, respectively. We also identified a sex-specific upregulation of genes associated with negative regulation of mRNA processing and an increase in intron retention consistent with a reduction in mRNA splicing in XY testicular GCs by E13.5. Using computational gene regulation network inference analysis, we identified sex-specific, sequential waves of putative key regulator genes during GC differentiation and revealed that the meiotic genes are regulated by positive and negative master modules acting in an antagonistic fashion. Finally, we found that rare adrenal GCs enter meiosis similarly to ovarian GCs but display altered expression of master genes controlling the female and male genetic programs, indicating that the somatic environment is important for GC function. Our data is available on a web platform and provides a molecular roadmap of GC sex determination at single-cell resolution, which will serve as a valuable resource for future studies of gonad development, function and disease.

To investigate the role of the nuclear receptor NR5A1 in the testis after sex determination, we analyzed mice lacking NR5A1 in Sertoli cells (SCs) from embryonic day (E) 13.5 onwards. Ablation of Nr5a1 impaired the expression of genes characteristic of SC identity (e.g. Sox9 and Amh), caused SC death from E14.5 onwards through a Trp53-independent mechanism related to anoikis, and induced disorganization of the testis cords. Together, these effects caused germ cells to enter meiosis and die. Single-cell RNA-sequencing experiments revealed that NR5A1-deficient SCs changed their molecular identity: some acquired a ‘pre-granulosa-like’ cell identity, whereas other reverted to a ‘supporting progenitor-like’ cell identity, most of them being ‘intersex’ because they expressed both testicular and ovarian genes. Fetal Leydig cells (LCs) did not display significant changes, indicating that SCs are not required beyond E14.5 for their emergence or maintenance. In contrast, adult LCs were absent from postnatal testes. In addition, adult mutant males displayed persistence of Müllerian duct derivatives, decreased anogenital distance and reduced penis length, which could be explained by the loss of AMH and testosterone synthesis due to SC failure.

DND1 is essential to maintain germ cell identity. Loss ofDnd1function results in germ cell differentiation to teratomas in some inbred strains of mice or to somatic fates in zebrafish. Using our knock-in mouse line in which a functional fusion protein between DND1 and GFP is expressed from the endogenous locus (Dnd1^GFP), we distinguished two male germ cell (MGC) populations during late gestation cell cycle arrest (G0), consistent with recent reports of heterogeneity among MGCs. Most MGCs express lower levels of DND1-GFP (DND1-GFP-lo), but some MGCs express elevated levels of DND1-GFP (DND1-GFP-hi). A RNA-seq time course confirmed highDnd1transcript levels in DND1-GFP-hi cells along with 5-10-fold higher levels for multiple epigenetic regulators. Using antibodies against DND1-GFP for RNA immunoprecipitation (RIP)-sequencing, we identified multiple epigenetic and translational regulators that are binding targets of DND1 during G0 including DNA methyltransferases (Dnmts), histone deacetylases (Hdacs), Tudor domain proteins (Tdrds), actin dependent regulators (Smarcs), and a group of ribosomal and Golgi proteins. These data suggest that in DND1-GFP-hi cells, DND1 hosts coordinating mRNA regulons that consist of functionally related and localized groups of epigenetic enzymes and translational components.