Single-nucleus multiomics of murine gonads reveals gene-regulatory networks underlying sex determination

Sex determination in the mammalian gonad involves coordinated lineage decisions in both germ cells and gonadal supporting cells. To define the regulatory programs driving these processes, we generated a single-nucleus multiomics atlas of murine fetal gonads, integrating chromatin accessibility and gene expression from individual nuclei across sex determination timepoints in both XX and XY gonads. In primordial germ cells (PGCs), joint RNA- and ATAC-seq profiling identified transcriptional states and regulatory elements underlying germ cell sex determination. These data resolved previously uncharacterized PGC subpopulations, revealed that changes in chromatin accessibility often precede transcriptional activation during PGC lineage commitment, and showed that sexual dimorphism in gene expression and chromatin state increases over developmental time. Motif enrichment and gene-regulatory network analyses highlighted distinct cohorts of transcription factors associated with XX and XY germ cell differentiation, including TFAP2C, TCFL5, GATA2, MGA, NR6A1, TBX4, and ZFX in XX PGCs, and forkhead-box and POU6 factors in XY PGCs. The dataset also captured dynamic signaling pathways (WNT, BMP, and RA) and potential cell-cell communication interactions between PGCs and the surrounding somatic niche. In the supporting cell lineage, integrated snRNA- and snATAC-seq analyses revealed how precursor cells diverge to form Sertoli cells in testes and granulosa cells in ovaries. Sex-specific chromatin accessibility in supporting cells emerged shortly after sex determination, accompanied by activation-associated histone marks. Motif analyses identified regulatory networks associated with ovary-enriched transcription factors such as LEF1 and MSX1, which promote granulosa fate by activating key target genes including Foxl2 and Fst (bioRxiv 2025.02.24.639877). Together, this dataset provides a multimodal reference map of the chromatin landscapes, transcriptional programs, and regulatory networks that coordinately govern sex determination of germ cells and supporting cells during murine gonadal development.