09-P060 Evaluation of acridine orange as a marker of germ cell developmental stages in the developing zebrafish ovary

molecular mechanism(s) of the regulation of Otx2 expression remains to be elucidated. We previously identified the cis-element that drives Otx2 expression in the forebrain and midbrain from E8.5, termed FM enhancer. In this study, we showed that the 157-bp core sequence in the FM enhancer was sufficient for the activation of reporter gene expression in the forebrain and midbrain. The core sequence contains the Tcf/Lef and Otx binding sites, which are deeply conserved among vertebrates. Physical interactions of Lef1 and Otx2 to these sites were observed by EMSA. Furthermore, mutations introduced in these sites caused reduction of the FM enhancer activity in the transient reporter analysis. We also demonstrated that another conserved 29-bp region in the 157-bp FM enhancer is necessary for the enhancer activity. By DNA affinity purification followed by mass spectrometry analyses, it was shown that classIII POU factors Brn1/2/4 interact with the 29-bp region. The homeodomain recognition site (TAATTA) in this region was shown to be necessary for the binding of the Brn1/2/4 invitro. Supershift analysis using antiBrn2 antibody suggested the binding of endogenous Brn2 to this region. From these results, it is inferred that brain regionalization is controlled by the orchestration of these transcription factors on the FM enhancer through regulating Otx2 expression.