The Pou5f1/Pou3f-dependent but SoxB-independent regulation of conserved enhancer N2 initiates Sox2 expression during epiblast to neural plate stages in vertebrates

► Sox2 expression in epiblast and ANP is regulated by enhancer N2. ► N2 activity depends on conserved POU binding motifs, but does not involve Sox2. ► Activating POU factors change from Pou5f1 to Pou3fs as ANP develops from epiblast. ► Zebrafish pou5f1 mutants fail to activate enhancer N2 and Sox2 in blastoderm/ ANP. ► Sox2 enhancer SRR2, requiring Pou5f1 and Sox2, is only active in amniote ES cells.