Figure 1

Induction of gExEn cells from ES cells by inducible Gata6 or Gata4 expression systems. (A-D) Morphology of differentiated SKG612 (A, B) and EBRTc-G6 (C, D) cells induced by withdrawal of Tc on gelatinized dishes (A, C) or under MEF culture conditions for 5 days (B, D). (E, F) Confocal microscopic image of ES cells with introduced pCAG-EGFP-G6GR 2 hr after the addition of 70% EtOH, without (E) and with (F) Dex. The left panels show phase-contrast images, the center panels show localization of EGFP-G6GR monitored by EGFP fluorescence, and the right panels show nuclei stained with Hoechst33342. (G-J) Morphology of 5G6GR11-2 ES cells with or without Dex. 5G6GR11-2 ES cells were grown in the presence (G) or absence (H) of LIF for 5 days, or were treated with 100 nM Dex for 5 days on gelatinized dishes (I, high-density conditions in inset) or on MEF (J). (K) Expression levels of endogenous Gata6 and Gata4 in ES-derived ExEn cells 4 days after activation of exogenous Gata on gelatinized dish cultures or PrE induced by withdrawal of LIF for 5 days. XEN2 and XEN9 cells; derived from embryo and MEF-dependent propagation. MEF; MEF alone. All values were normalized relative to the level of Gapdh and plotted relative to levels of expression in XEN2 cells. (L) Expression levels of endogenous Fgf3 in ES-derived ExEn cells 4 days after activation of exogenous GATA on gelatinized dish cultures, or PrE induced by withdrawal of LIF for 5 days. XEN2 and XEN9 cells; derived form embryo and MEF-dependent propagation. MEF; MEF alone. All values were normalized relative to the level of Gapdh and plotted relative to levels of expression in XEN2 cells. (M) GATA-dependent enhancer activity of the element containing GATA-binding site of pFgf3-luc in ES-derived ExEn cells. Reporter plasmids were transfected into 5G6GR11-2 and 5G4GR4-3 ES cells followed by culture with (exo-GATA ON) or without Dex (OFF), or EBRTc-G6 and SKG612 ES cells followed by culture with (OFF), or without Tc (exo-GATA ON). Relative expression levels of pFgf3-luc (filled; exo-GATA ON, hatched; OFF) are shown. All results were normalized relative to the luciferase activities of pCMV-RL and plotted relative to the luciferase activities of pGL3-luc in Dex-non-treated 5G6GR11-2, set at 1.0.