Proper formation of germ cells during embryonic development is crucial for ensuring the production of functional gametes (McLaren, 2003). The embryonic precursors to mature gametes, primordial germ cells (PGCs), are the bipotential stem cells of the germline that give rise to eggs and sperm (McLaren, 2003; Hancock et al., 2021). During embryonic development in mice, PGCs commit to the oogenic or spermatogenic lineage in response to sex-determining cues from the gonadal environment in a process termed PGC sex determination (Spiller and Bowles, 2015). During sex determination, XX PGCs in the fetal ovary commit to oogenesis by entering meiosis immediately after pre-granulosa cell specification (Borum, 1961). By contrast, XY PGCs in the fetal testis initiate the spermatogenic program and arrest mitotically in response to signals from Sertoli cells (Spiller and Bowles, 2015; Bowles and Koopman, 2010). Defects in germ cell differentiation, particularly during fetal life, often lead to reproductive diseases, such as infertility (Czukiewska and Chuva de Sousa Lopes, 2022) and the formation of germ cell tumors (Oosterhuis and Looijenga, 2019). Thus, it is necessary to enhance our understanding of germ cell development so we can better identify the etiologies of reproductive dysfunction in humans.
PGC sex determination is induced by the sex-specific activation of transcription factors (TFs) and downstream gene networks (Spiller and Bowles, 2022). In XX PGCs, the retinoic acid (RA)-responsive TFs STRA8 and MEIOSIN and the bone morphogenetic protein (BMP)-responsive TF ZGLP1 are required for entry into meiosis and oogenesis (Spiller and Bowles, 2022; Ishiguro et al., 2020; Nagaoka et al., 2020). These TFs also initiate the expression of genes related to meiotic processes, including Rec8 and Sycp1-3 (Spiller and Bowles, 2022). In contrast, XY PGCs require the expression of cell cycle inhibitors, such as Bnc2 and Cdkn2b, and the male-specific genes, Nanos2 and Dnd1, to enter mitotic arrest (Vanhoutteghem et al., 2014; Spiller et al., 2010; Saba et al., 2014; Cook et al., 2011). During the transition from PGC to oogonium or gonocyte, both XX and XY PGCs must also lose their bipotential state by downregulating the pluripotency-related genes, Pou5f1, Nanog, and Sox2 (Spiller and Bowles, 2022). Consequently, there are multiple layers of gene regulation required for sex determination of PGCs.
Beyond the patterns of gene expression in PGCs, relatively little is known about how signals from the gonadal environment activate the expression of sexually dimorphic TFs and genes in PGCs. First, the gene regulatory networks, i.e., TFs and their predicted target genes, specific to XX and XY PGCs are not well defined. Second, it remains unclear how the chromatin environment is temporally regulated during PGC sex determination. Finally, additional data are needed on the patterns of ligand-receptor expression in gonadal supporting cells and PGCs. Previous reports have used bulk gene regulation and expression genomics assays to investigate the transcriptional programs underlying PGC development (Houmard et al., 2009; Jameson et al., 2012; Lesch et al., 2013; Rolland et al., 2011). However, these assays may not have the resolution or sensitivity required to detect the transient changes in gene regulation among PGC subpopulations that are essential to sex determination.
In the present study, we employed the integrative genomics method, combined single-nucleus transcriptome and chromatin accessibility sequencing from the same cell, to decipher various layers of gene regulation during PGC sex determination in mice. We comprehensively profiled 3,054 XX and XY PGCs at embryonic days (E) E11.5, E12.5, and E13.5, which covers the developmental time frame from bipotential to sexually differentiated PGCs. Single-nucleus sequencing enabled the detection of sex-enriched regulatory loci, TFs, and gonadal cues that may be responsible for initiating gene expression in individual PGCs. We first systematically examined the molecular signatures of PGC subpopulations to identify the genes and accessible chromatin regions underpinning the sex-specific fates of PGCs. By combining our epigenomic and transcriptomic data, we predicted cis-regulatory elements and sex-enriched TFs to construct the gene regulatory networks unique to XX and XY PGCs. Lastly, we probed the cell-cell communication pathways between supporting cells and PGCs to nominate potentially new ligand-receptor pairs involved in PGC development. Our results provide insights into the cell fate decisions underlying gametogenesis and sex determination of PGCs.