GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program [Bisulfite-Seq]

The in vitro reconstitution of human germ-cell development provides a robust framework for clarifying key underlying mechanisms. Here, we explored transcription factors (TFs) that engender the germ-cell fate in their pluripotent precursors. Unexpectedly, SOX17, TFAP2C, and BLIMP1, which act under the BMP signaling and are indispensable for human primordial germ-cell-like cell (hPGCLC) specification, failed to induce hPGCLCs. In contrast, GATA3 or GATA2, immediate BMP effectors, combined with SOX17 and TFAP2C, generated hPGCLCs. GATA3/GATA2 knockouts dose-dependently impaired BMP-induced hPGCLC specification, whereas GATA3/GATA2 expression remained unaffected in SOX17, TFAP2C, or BLIMP1 knockouts. In cynomolgus monkeys, a key model for human development, GATA3, SOX17, and TFAP2C were co-expressed exclusively in early PGCs. Crucially, the TF-induced hPGCLCs acquired a hallmark of bona fide hPGCs to undergo epigenetic reprogramming and mature into oogonia/gonocytes in xenogeneic reconstituted ovaries. By uncovering a TF circuitry driving the germ line program, our study provides a paradigm for TF-based human gametogenesis. Whole Genome Bisulfite Sequence (WGBS): Overexpression of SOX17, TFAP2C, GATA3, clone_1; day77

人类生殖细胞发育的体外重构体系，为阐明其核心潜在机制提供了坚实的研究框架。本研究旨在探索可在多能前体细胞中赋予其生殖细胞命运的转录因子（transcription factors, TFs）。令人意外的是，在骨形态发生蛋白（bone morphogenetic protein, BMP）信号通路下游发挥作用、且对人类原始生殖细胞样细胞（human primordial germ-cell-like cell, hPGCLC）特化过程不可或缺的SOX17、TFAP2C与BLIMP1，却无法诱导产生人类原始生殖细胞样细胞。与之相反，作为BMP信号直接效应分子的GATA3或GATA2，与SOX17和TFAP2C联合使用时，可成功诱导产生人类原始生殖细胞样细胞。GATA3与GATA2的基因敲除会以剂量依赖性方式削弱BMP诱导的hPGCLC特化过程；而在SOX17、TFAP2C或BLIMP1基因敲除的样本中，GATA3与GATA2的表达并未受到影响。作为人类发育研究关键模型的食蟹猴体内，GATA3、SOX17与TFAP2C仅在早期原始生殖细胞（primordial germ cells, PGCs）中共表达。至关重要的是，经转录因子诱导产生的人类原始生殖细胞样细胞，具备真实人类原始生殖细胞的标志性特征：可发生表观遗传重编程，并在异种重构卵巢中成熟为卵原细胞/性原细胞。本研究揭示了驱动生殖系程序的转录因子调控网络，为基于转录因子的人类配子发生研究提供了范式。全基因组亚硫酸氢盐测序（Whole Genome Bisulfite Sequence, WGBS）：SOX17、TFAP2C、GATA3过表达组，克隆1；培养第77天