Figure 5. Dose-dependent function of GATA transcription factors in human primordial germ-cell-like cell specification.

(A) Bright-field and fluorescence (AG/BT) images, and FACS analyses for BTAG expression in floating aggregates of the GATA3^−/−, GATA2^−/−, and parental clones induced by bone morphogenetic protein 4 (BMP4) at d2/d4. Representative images of at least two independent experiments are shown (indicated in Fig 5F). Bars, 200 μm. (B) Expression dynamics of the indicated genes during human primordial germ-cell–like cell induction (iMeLCs, d2/d4 BT⁺AG⁺ cells) by BMP4 from the parental (black), GATA3^−/− (red), and GATA2^−/− (blue) clones. For each gene, the ΔCt was calculated from the average Ct value of two housekeeping genes, RPLP0 and PPIA (set as 0). The bars indicate the mean value of each time point of each genotype. Replicate numbers: GATA2^−/−: 3; GATA3^−/−: 4 for iMeLCs and 8 for d2/d4 BT⁺AG⁺ cells; parental clone: 2. (C, D) The principal component analysis plots of the cells (iMeLCs, d2/d4/d6 BT⁺AG⁺ cells) derived from the GATA3^−/− (C) and GATA2^−/− (D) clones (squares), overlaid with the indicated cells derived from the parental clone (circles with pale colors). See Table S1 for the samples analyzed. The color coding is as indicated. (E) Bright-field and fluorescence (AG/BT) images, and FACS analyses for BTAG expression in floating aggregates of the GATA3^−/−; GATA2^+/− and GATA3^−/−; GATA2^−/− clones induced by BMP4 at d4. Representative images of at least two independent experiments are shown (indicated in Fig 5F). Bars, 200 μm. (F) The percentages of BT⁺AG⁺ cell induction from the indicated genotypes at d4. The replicate numbers and the P-values (t test) are as indicated. The inductions were performed side by side. Typically, the efficiency for BT⁺AG⁺ cell induction from parental hiPSCs varies to this extent (20%∼60%) (9, 13, 26). (G) A scheme for GATA3 expression in the GATA3^−/−; GATA2^−/−; GATA3 clone. (H) Bright-field and fluorescence (AG/BT) images, and FACS analyses for BTAG expression in floating aggregates of the GATA3^−/−; GATA2^−/−; GATA3 clone upon induction with BMP4 and 0, 0.5, and 1.0 μg/ml of Dox at d4. Representative images of at least two independent experiments are shown (indicated in Fig 5F). Bars, 200 μm. (I) The percentages of BT⁺AG⁺ cell induction at d4 (two replicates) from the GATA3^−/−; GATA2^−/−; GATA3 clone induced with BMP4 and 0, 0.5, and 1.0 μg/ml of Dox treatment. (J) Expression of the indicated genes in d4 BT⁺AG⁺ cells induced from the parental (with BMP4, red) or GATA3^−/−; GATA2^−/−; GATA3 (with BMP4 and Dox, green) clones (two replicates). For each gene, the ΔCt was calculated from the average Ct value of two housekeeping genes, RPLP0 and PPIA (set as 0).