Differentiation of embryonic stem cells is induced by GATA factors

Abstract

Extraembryonic endoderm (ExE) is differentiated from the inner cell mass of the late blastocyst-stage embryo to form visceral and parietal endoderm, both of which have an important role in early embryogenesis. The essential roles of Gata-6 and Gata-4 on differentiation of visceral endoderm have been identified by analyses of knockout mice. Here we report that forced expression of either Gata-6 or Gata-4 in embryonic stem (ES) cells is sufficient to induce the proper differentiation program towards ExE. We believe that this is the first report of a physiological differentiation event induced by the ectopic expression of a transcription factor in ES cells.

Figure 1

Supertransfection of Gata-4 and Gata-6 expression vector into MG 1.19 embryonic stem (ES) cells. (A) Photographs of colonies 7 d after transfection with each expression vector and selected in the presence of leukemia inhibitory factor (LIF). Gata-6 and Gata-4 transfectants show similar PE like morphology. (B) Northern blot analysis of endoderm markers in total RNAs from undifferentiated (stem), empty, Gata-6, Gata-4, Oct-3/4, and Stat3F supertransfected MG 1.19 ES cells. Reciprocal transactivation between Gata-6 and Gata-4 results in endoderm marker gene expression. (C) RT–PCR analysis of germ layer markers. (D) Western blots of nuclear extracts from empty, Gata-6, and Gata-4 vector transfected MG 1.19 cells. Induced expression of Gata-6 protein results in Gata-4 protein expression, and vice versa.

Figure 2

Gata-6 overexpression in stem cells leads to endoderm differentiation and blocks stem cell states. (A) An inducible Gata-6 transgene integration by supertargeting. For diagnosis of the supertargeting event, the selected clones were analyzed by Southern blot hybridization as reported previously (Niwa et al. 1998). A 3.2-kb SacI fragment was detected with a probe from the 5′ end of lacZ, indicative of the replacement of the Oct-3/4 cDNA with Gata-6. Original ZHTc6 cells retained the 4.8-kb fragment of the preexisting Oct-3/4 transgene. Supertargeting events had taken place in all clones tested, and SKG612 is representing one of such clones. (B) Western blot analysis of SKG612 embyonic stem (ES) cells using anti-Gata-6 or anti-Gata-4 antibody after incubation for 24 h at the indicated concentrations of tetracycline (Tc). Induced expression of Gata-6 and Gata-4 protein was detected in SKG612 cells. (C) Colony morphology of SKG612 cells cultured for 4 d in the presence of leukemia inhibitory factor (LIF) and absence of Tc. (D) Electron micrograph of differentiated SKG612 cells. Magnification, ×4000. (E) Time course for the activation of endoderm associated genes and the repression of stem associated genes during the differentiation of SKG612 cells, analyzed by Northern blot. (F) Time course for the activation of endoderm associated genes and the repression of Bmp-4, analyzed by RT–PCR.

Figure 3

Gata-6 promotes terminal differentiation of extraembryonic endoderm (ExE) after withdrawal of leukemia inhibitory factor (LIF) and promotes visceral endoderm (VE) differentiation under aggregated cell culture. (A) Northern blot analysis of SKG612 cells after (I) 4 d culture in the presence of LIF and tetracycline (Tc), (II) 2 d culture in the absence of LIF and presence of Tc, (III) further 2 d (total 4 d) following the withdrawal of Tc from a culture that was grown for 2 d in the absence of LIF and presence of Tc, (IV) 4 d culture in the absence of LIF and the presence of Tc. Gata-6 works not only as initiator but also as promoter of endoderm differentiation. (B) Photographs of SKG612 cells cultured under conditions described above. (C) SKG612 cells were cultured in suspension for 0, 2, 4, or 6 d in the presence of LIF and in the presence or absence of Tc. RNA was isolated at each time point and analyzed by RT–PCR. Note the correlation between the expression of endoderm-associated genes and concurrent or prior expression of Gata-6. (D) Electron micrograph of differentiated SKG612 cells in aggregated culture. Magnification, ×6000.

Figure 4

Forced expression of Coup-tf I represses stem cell marker genes. (A) Northern blot analysis of stem cell and differentiation marker genes in total RNAs from MG1.19 embryonic stem (ES) cells supertransfected with empty or Coup-tf I expression vectors. Overexpression of Coup-tf I results in down-regulation of stem cell marker genes Oct-3/4 and Utf-1. (B) Colony morphology of Coup-tf I supertransfectants. These cells show epithelial-like morphology distinct from extraembryonic endoderm (ExE). (C) Model for early endoderm differentiation of ES cells. See the text for details.