Development of the anterior forebrain precursor (AFBP) in the anterior neural 
plate (ANP) depends on the activation of the Hesx1 transcription factor gene. 
The Hesx1-expression domain of the ANP is underlain by Dkk1-expressing tissues, 
initially proximal-most anterior visceral endoderm (AVE), and later anterior 
mesendoderm (AME). As Dkk1-null embryos fail to develop the Hesx1-expressing 
domain, it is likely that Wnt signal inhibition in the ANP is required for the 
Hesx1 activation. To investigate the regulation of the AFBP development, we took 
advantage of epiblast stem cells (EpiSCs), which develop into the ANP in the 
absence of activin signaling. Expression of Hesx1 and Six3, both involved in the 
AFBP development, was strongly activated 2 days after activin removal and 
concomitant addition of Wnt signal inhibitors, Dkk1 or XAV939. Furthermore, we 
showed that activation of the 720-bp Hesx1 5' enhancer is responsible for Hesx1 
expression in the AFBP and depends on Wnt signal inhibition. In addition, we 
showed that Wnt inhibition during the first day has larger impact on the 
activation of Hesx1 and Six3 than the second day, suggesting that in embryos Wnt 
inhibition caused by the AVE-derived Dkk1, rather than the AME-derived Dkk1, 
contributes greatly in the establishment of the AFBP.