Dosage-dependent role of Gata3 in primitive endoderm and trophectoderm specification in embryonic stem cells [RNA-seq]

Transcription factors (TFs) are critical regulators of cell fate decisions, and their intricate balance determines lineage specification during early development. Gata3 is a well-established TF in the trophectoderm (TE) development, and its ectopic expression can reprogram mouse embryonic stem (ES) cells into trophoblast stem (TS) cells. Because of Gata3’s canonical role in TE, its potential involvement in primitive endoderm (PE) differentiation has remained unexamined, representing a previously overlooked avenue of investigation. Here, we reveal that Gata3 functions as a dosage-sensitive switch governing lineage commitment in mouse ES cells. Modulating Gata3 expression revealed that high levels (Gata3-H) drive TE specification, whereas lower levels (Gata3-L) promote PE differentiation, as evidenced by distinct morphological transitions, transcriptional profiles, chromatin-binding, and functional assays. Mapping targets of Gata3 revealed differential occupancy patterns at PE- versus TE-associated enhancers correlating with dose. In vitro 3D blastoid models confirmed that Gata3 dosage alone is sufficient to drive spatial segregation, with Gata3-L and Gata3-H cells preferentially localizing to PE- and TE-like compartments, respectively. Our findings highlight Gata3’s pioneer factor-like behavior, downregulation of pluripotency networks, and latent capacity to direct lineage specification beyond its classical TE specification. These results expand the understanding of Gata family TFs in early embryogenesis and provide mechanistic insights for stem-cell based models for lineage specification.