Regulation of Sox2 in the pre-placodal cephalic ectoderm and central nervous system by enhancer N-4

The development of various tissues originating from the cephalic placodes is accompanied by the expression of the Sox2 gene. This Sox2 expression initiates in the pre-placodal cephalic ectoderm, and is regulated by enhancer N-4, which also regulates Sox2 in the embryonic central nervous system (CNS) posterior to the diencephalon. As the regulation of enhancer N-4 in the ectoderm likely reflects that of the pre-placodal cell state, its regulatory elements were characterized. A 110-bp minimal and essential sequence of N-4 (mini-N-4) was determined. By mutational and deletion analyses, nine regulatory elements were determined in the mini-N-4 sequence: three elements involved in activation in both the cephalic ectoderm and CNS, three elements specifically involved in activation in the cephalic ectoderm, three elements individually involved in activation in the mesencephalon, repression in the prosencephalon, and retinoic acid response in the rhombomeric region. The cephalic ectoderm-specific elements include two potential sites for the binding of nuclear receptors, suggestive of a nuclear receptor-dependent regulation. Multimers of the 3' half of the mini-N-4 sequence, including all of the cephalic ectodermal elements, show strong and selective activity in the cephalic ectoderm, providing a powerful genetic tool for the manipulation of gene activities in the placodal lineages.