Fig 1: GATA3 Is Necessary for Timely Differentiation and Can Drive Differentiation in the Absence of BMP4 Signals(A) Quantification of GATA3 expression during 3 days of BMP4 (50 ng/mL) treatment.(B) Left: representative images of GATA3 and NANOG expression after BMP4 (50 ng/mL) treatment. Scale bar represents 100 µm. Center: GATA3 and NANOG expression in space after BMP4 stimulation, assuming a spherical geometry for hESC colonies. Right: quantification of GATA3 and NANOG levels in single cells after BMP4 treatment. More than 10,000 cells were analyzed for each experimental condition. n = 3 independent experiments.(C) Quantification of GATA3 ChIP-qPCR in the presence (red) or absence (blue) of BMP4 (50 ng/mL) treatment of hESC (for 6 h). BMP4, HES1, CDX2, and SNAI2 are genes shown to be involved in hESC differentiation. ISL1 and FOXC1 are lateral mesoderm (cardiac)-specific genes. A primer set -3 kb from BMP4 was used as a negative control (Neg control). Error bars represent means ± SDs for n = 2 biological replicates (**p < 0.001, ***p < 0.0001, ns is not significant).(D) Quantification of fold change of pluripotency (blue) and lineage-specific (red) genes in response to BMP4 stimulation (left) or tamoxifen-induced GATA3 expression (iGATA3) (right) relative to untreated control cells. The housekeeping gene GUSB was used for normalization. Error bars represent ±SDs from n = 3 biological replicates.(E) Quantification of the fraction of positive cells for pluripotency gene NANOG and lineage-specific markers BRY, SOX17, and CDX2 in response to BMP4 (50 ng/mL) stimulation in GATA3-/- (GATA3 KO) cells (red) or control cells (blue). n > 500 cells were analyzed for each experimental condition.(F) scRNA-seq data of wild type (WT) and GATA3 KO hESC stimulated with BMP4 for 48 h. Combined cell transcriptomes were analyzed with a mean gene per cell value of ~6,500 and a total read depth of ~600 million reads per cell. Cell transcriptomes were projected onto a diffusion map with shared 14 nearest-neighbor clusters highlighted (first 30 dimensions, resolution 0.8).(G) Heatmaps showing normalized counts per cell for GATA3 and mesoderm differentiation genes for WT (left) and GATA3 KO (right) hESCs after 48 h BMP4 stimulation. The left panel highlights a gradient of GATA3 expression.