One of the earliest epithelial-to-mesenchymal transitions in mouse embryogenesis 
involves the differentiation of inner cell mass cells into primitive and then 
into parietal endoderm. These processes can be recapitulated in vitro using F9 
teratocarcinoma cells, which differentiate into primitive endoderm when treated 
with retinoic acid (RA) and into parietal endoderm with subsequent treatment 
with dibutyryl cyclic adenosine monophosphate (db-cAMP). Our previous work on 
how primitive endoderm develops revealed that the Wnt6 gene is upregulated by 
RA, leading to the activation of the canonical WNT-β-catenin pathway. The 
mechanism by which Wnt6 is regulated was not determined, but in silico analysis 
of the human WNT6 promoter region had suggested that the GATA6 and FOXA2 
transcription factors might be involved [1]. Subsequent analysis determined that 
both Gata6 and Foxa2 mRNA are upregulated in F9 cells treated with RA or RA and 
db-cAMP. More specifically, overexpression of Gata6 or Foxa2 alone induced 
molecular and morphological markers of primitive endoderm, which occurred 
concomitantly with the upregulation of the Wnt6 gene. Gata6- or 
Foxa2-overexpressing cells were also found to have increased levels in T-cell 
factor (TCF)-dependent transcription, and when these cells were treated with 
db-cAMP, they developed into parietal endoderm. Chromatin immunoprecipitation 
analysis revealed that GATA6 and FOXA2 were bound to the Wnt6 promoter, and 
overexpression studies showed that these transcription factors were sufficient 
to switch on the gene expression of a Wnt6 reporter construct. Together, these 
results provide evidence for the direct regulation of Wnt6 that leads to the 
activation of the canonical WNT-β-catenin pathway and subsequent induction of 
primitive extraembryonic endoderm.