The homeobox gene Nanog is a key intrinsic determinant of self renewal in 
embryonic stem (ES) cells, and its repression leads ES cells to selectively 
differentiate into primitive endoderm. Although Nanog repression occurs at the 
outermost layer of ES cell aggregates independent of the leukemia inhibitory 
factor (LIF)/STAT3 pathway, it is largely undetermined what external cues and 
intracellular signals cause the event. Of interest, addition of the tyrosine 
phosphatase inhibitor, sodium vanadate, selectively repressed Nanog 
transcription without any detectable changes in upstream transcriptional 
regulators Oct3/4 and Sox2. Furthermore, sodium vanadate induced primitive 
endoderm differentiation, even in the inner cells of ES cell aggregates. 
Expression of Gata6 and Zfp42, two putative downstream Nanog effectors, was also 
increased and decreased by the addition of sodium vanadate, respectively, but 
these changes were eliminated by exogenous Nanog expression. The effects of 
sodium vanadate were abrogated by Grb2 deficiency or by the addition of the Mek 
inhibitor, PD98059. Indeed, PD98059 prevented Nanog repression induced by ES 
cell aggregation as well. Furthermore, transfection of a constitutive active Mek 
mutant into ES cells induced Nanog repression and primitive endoderm 
differentiation. These data indicate that the Grb2/Mek pathway primarily 
mediates Nanog gene repression upon ES cell differentiation into primitive 
endoderm.