GATA transcription factors, SOX17 and TFAP2C, drive the human germ-cell specification program

Abstract

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.

Expanding homogeneous culture of human primordial germ cell-like cells maintaining germline features without serum or feeder layers

“…for in vivo NANOG + /NANOS3 + hPGCs in a later stage at 4 weeks of gestation ( Li et al., 2017 ) expressed the late-PGC markers DAZL and DDX4 weakly but at unignorable levels ( Figures S6 C–S6E). In either of these single-cell RNA-seq data, expression of GATA2 or GATA3 was not detected in hPGCs ( Figures S6 B, S6D, and S6E), placing our c0 hPGCLCs to a unique GATA2 + /GATA3 + stage in hPGC development as described by Kojima et al. (2021) .…”

“…On the other hand, single-cell RNA-seq data presented by Li et al for in vivo NANOG + / NANOS3 + hPGCs in a later stage at 4 weeks of gestation (Li et al, 2017) expressed the late-PGC markers DAZL and DDX4 weakly but at unignorable levels (Figures S6C-S6E). In either of these single-cell RNA-seq data, expression of GATA2 or GATA3 was not detected in hPGCs (Figures S6B, S6D, and S6E), placing our c0 hPGCLCs to a unique GATA2 + /GATA3 + stage in hPGC development as described by Kojima et al (2021). Taken together, these results indicate that hPGCLCs experience significant transcriptomic changes during the initial transition from the GATA2/3positive pre-expansion state to the GATA2/3-negative post-expansion state, whereas both the pre-and postexpansion cells are legitimate hPGCLCs.…”

“…Prolonged expression of GATA3 from an inducible vector in hPGCLCs in the BMP4-containing hPGCLC induction medium resulted in major cell death (Kojima et al, 2021). Thus, our GATA2/3-positive, freshly isolated (c0) hPGCLCs correspond with the GATA2/3-positive hPGCLCs observed by others (Chen et al, 2019;Kojima et al, 2021;Murase et al, 2020), and our GATA2/ 3-negative LTC-hPGCLCs are equivalent to the post-expansion hPGCLCs described by Murase et al (2020). Importantly, both the pre-and the post-expansion hPGCLCs obtained by us and others expressed the key hPGC (LC) markers, indicating that the two states of cells are both legitimate hPGCLCs.…”

Germline specification from pluripotent stem cells

“…The disruption of SOX17 caused the failure of hPGC specification, while the overexpression of SOX17 was able to improve the efficiency of hPGC specification [ 21 ]. In addition, GATA3 or GATA2 as the immediate BMP effectors, combined with SOX17 and TFAP2C was able to promote hPGCLCs generation [ 56 ]. EOMES encodes many T-box-containing TFs and is required for the development of extraembryonic ectoderm in mouse post-implantation embryos and for gastrulation [ 57 ].…”

In silico discovery of small molecules for efficient stem cell differentiation into definitive endoderm

“…1B). However, the role of these markers in endoderm formation is not yet understood, although recent studies suggest that they might be involved in the process (Kojima et al 2021;Li et al 2019). For ME, we identified the following activated TFs: CDX1/2/4, which are involved in hematopoiesis (Paik et al 2013), TCF4 (Kardon, Harfe, and Tabin 2003), LEF1 (Galceran et al 2004), SP5 (Weidinger et al 2005), HOXB (Iimura and Pourquié 2006), TBX6 (Sadahiro et al 2018), and FOXH1 (David and Massagué 2018), which is also involved in heart development (von Both et al 2004).…”