INDUCTION OF YOLK-SAC ENDODERM IN GATA-4-DEFICIENT EMBRYOID BODIES BYRETINOIC ACID

GATA-4, a transcription factor implicated in lineage determination, is expressed in both parietal and visceral endoderm of the early mouse e mbryo. In embryonic stem cell-derived embryoid bodies, GATA-4 mRNA is first detectable at 4-5 days of differentiation and is confined to vis ceral endoderm cells on the surface of the bodies. Previously we repor ted that targeted mutagenesis of the Gata4 gene in embryonic stem cell s results in a block in visceral endoderm differentiation in vitro. In an attempt to elucidate the role of GATA-4 in the formation of viscer al endoderm, we have now differentiated Gata4 -/- and wild type embryo id bodies in the presence of retinoic acid +/- dbcAMP, known inducers of endoderm formation. We show that differentiation of Gata4 -/- embry oid bodies in the presence of retinoic acid results in formation of vi sceral endoderm, while differentiation of Gata4 -/- embryoid bodies in the presence of retinoic acid plus dbcAMP causes parietal endoderm fo rmation. The presence of these yolk sac endoderm layers was confirmed by light microscopy and analysis of biochemical markers including ol-f etoprotein, type IV collagen, laminin, and binding sites for Dolichos biflorus agglutinin. Treatment of Gata4 -/- embryoid bodies with retin oic acid induces expression of another GATA-binding protein, GATA-6, i n both visceral and parietal endoderm cells. That another GATA-binding protein is induced in the absence of GATA-4 suggests that this family of transcription factors plays an important role in yolk sac differen tiation. (C) 1997 Elsevier Science Ireland Ltd.