Somites provide the basic body plan for metameric axial structures in vertebrates, and establish the segmental features through the sequential gene expression in the presomitic mesoderm (PSM). A crucial protein for segment border formation is the bHLH transcription factor Mesp2, the expression of which is restricted to the anterior PSM. A gene candidate that is activated by Mesp2 is Epha4, as its expression pattern resembles Mesp2 and is absent in Mesp2-null embryos. We have analyzed the enhancer region of Epha4, which is responsible for its expression in the anterior PSM, and identified an E-box containing region. Subsequent transgenic and transient luciferase analyses successfully determined that the presence of repeated E-box sequences is a minimum essential requirement for the expression in the anterior PSM. We also show that Mesp2 directly binds to the enhancer sequence of Epha4. Furthermore, the forced expression of Mesp2 in somitic cells results in the activation of Epha4 and repression of the caudal gene Uncx4.1, which may trigger the events leading to the formation of abnormal somites and rostralized vertebra. In addition, ectopic Mesp2 expression induces abnormally epithelialized structures, which support to the idea that Mesp2 induces the formation of segmental borders by activating genes that play roles in cellular epithelialization.