Single-nucleus multiomics reveals the gene-regulatory networks underlying sex determination of murine primordial germ cells

Accurate specification of female and male germ cells during embryonic development is critical for sexual reproduction. Primordial germ cells (PGCs) are the bipotential precursors of mature gametes that commit to an oogenic or spermatogenic fate in response to sex-determining cues from the fetal gonad. The critical processes required for PGCs to integrate and respond to signals from the somatic environment in gonads are not understood. In this study, we developed the first single-nucleus multiomics map of chromatin accessibility and gene expression during murine PGC development in both XX and XY embryos. Profiling of cell-type specific transcriptomes and regions of open chromatin from the same cell captured the molecular signatures and gene networks underlying PGC sex determination. Joint RNA and ATAC data for single PGCs resolved previously unreported PGC subpopulations and cataloged a multimodal reference atlas of differentiating PGC clusters. We discovered that regulatory element accessibility precedes gene expression during PGC development, suggesting that changes in chromatin accessibility may prime PGC lineage commitment prior to differentiation. Similarly, we found that sexual dimorphism in chromatin accessibility and gene expression increased temporally in PGCs. Combining single-nucleus sequencing data, we computationally mapped the cohort of transcription factors that regulate the expression of sexually dimorphic genes in PGCs. For example, the gene regulatory networks of XX PGCs are enriched for the transcription factors, TFAP2c, TCFL5, GATA2, MGA, NR6A1, TBX4, and ZFX. Sex-specific enrichment of the forkhead-box and POU6 families of transcription factors was also observed in XY PGCs. Finally, we determined the temporal expression patterns of WNT, BMP, and RA signaling during PGC sex determination, and our discovery analyses identified potentially new cell communication pathways between supporting cells and PGCs. Our results illustrate the diversity of factors involved in programming PGCs towards a sex-specific fate. Multiome-seq: Single nuclei were extracted from embryonic gonads from E11.5-E12.5 male and female mice. Joint snRNA and snATAC-seq libraries were prepared using the 10x Genomics platform.胚胎发育过程中雌性与雄性生殖细胞的精准特化，对于有性生殖至关重要。原始生殖细胞（Primordial Germ Cells, PGCs）是成熟配子的双潜能前体细胞，它们可响应胎儿性腺发出的性别决定信号，定向分化为卵母细胞发生或精子发生谱系。目前学界尚未明确PGCs整合并响应性腺体细胞微环境信号的关键过程。 本研究首次构建了小鼠XX和XY胚胎中PGC发育过程的染色质开放性（chromatin accessibility）与基因表达单细胞核多组学（single-nucleus multiomics）图谱。对同一细胞的细胞类型特异性转录组与开放染色质区域进行测序分析，揭示了PGC性别决定背后的分子特征与基因调控网络。针对单个PGC的联合RNA与ATAC测序（ATAC-seq）数据，解析了此前未被报道的PGC亚群，并构建了分化PGC簇的多模态参考图谱。 本研究发现，在PGC发育过程中，调控元件的开放性先于基因表达出现，这表明染色质开放性的改变或许可在分化发生前，为PGC的谱系定向奠定基础。此外，我们还发现PGC中染色质开放性与基因表达的性别二态性随时间推移逐渐增强。整合单细胞核测序数据，我们通过计算手段鉴定出了调控PGC内性别二态性基因表达的转录因子集合。例如，XX型PGC的基因调控网络中富集有TFAP2c、TCFL5、GATA2、MGA、NR6A1、TBX4及ZFX等转录因子。XY型PGC中也存在叉头框（forkhead-box）家族与POU6家族转录因子的性别特异性富集现象。 最后，我们明确了PGC性别决定过程中WNT、BMP及RA信号通路的时序表达模式，并通过探索性分析鉴定出了支持细胞与PGC之间潜在的新型细胞通讯通路。本研究结果揭示了参与PGC向性别特异性命运编程的各类因子的多样性。 多组测序（Multiome-seq）：从E11.5至E12.5阶段的雌雄小鼠胚胎性腺中提取单细胞核，采用10x Genomics平台构建联合单细胞核RNA测序（snRNA-seq）与单细胞核ATAC测序（snATAC-seq）文库。