Identification of a phylogenetically conserved activin-responsive enhancer in the Zic3 gene

Multiple signaling pathways are involved in the induction of the organizer, a major center controlling vertebrate body plan formation. To study these signals, we have focused on the regulation of the Zic3 gene, which codes for a zinc finger transcription factor expressed in the organizer region at the beginning of gastrulation. We searched for DNA regulatory elements in the Zic3 promoter by testing their ability to drive reporter gene expression in early embryos. By this approach, we identified an activin responsive enhancer (Zic3-ARE), which was located in the Zic3 first intron and was essential for dorsal activation of the reporter. The Zic3-ARE was stimulated by activin and Nodal ligands, but not by a dominant negative bone morphogenetic protein (BMP) receptor. The Zic3-ARE contains a repeating consensus homeodomain binding sequence, CTAATTAAA, suggesting involvement of a homeodomain transcription factor(s). Mutations in this motif abolished enhancer activity in dorsal marginal zone and its response to activin in animal pole explants. Inhibition of either Wnt/β-catenin or activin/Nodal signaling suppressed Zic3-ARE activity in dorsal blastomeres, further illustrating the importance of these pathways in activation of organizer genes.