The regulation of forkhead/HNf-3β expression in the Ciona embryo

The Ciona forkhead/HNF-3 beta gene (Ci-fkh) is expressed in the primary axial tissues of the developing tadpole, including the notochord, endoderm, and rudimentary boor plate of the CNS. In an effort to determine the basis for this complex pattern of expression we have conducted a detailed analysis of the Ci-Fh 5'-regulatory region. Different 5' sequences were attached to a lacZ reporter gene and analyzed in electroporated Ciona embryos. A short regulatory sequence (AS) located similar to1.7 kb upstream of the transcribed region is shown to be essential for expression in all three axial tissues. The proximal 20 bp of the AS contains overlapping Snail repressor elements and a T-box motif. Deleting these sequences causes the loss of reporter gene expression in the endoderm, as well as expanded expression in the neural tube. These results suggest that a T-box gene such as Ci-VegTR activates Ci-Fh expression in the endoderm, while the Ci-Sna repressor excludes expression from the lateral ependymal cells and restricts the Ci-Fh pattern to the rudimentary floor plate in ventral regions of the neural tube. We also present evidence for Ci-Fh positive autofeedback, whereby the Ci-Fkh protein binds to critical activator sites within the Ci-Fh 5'-regulatory region and helps maintain high levels of expression. We discuss these results with respect to forkhead/HNN-3 beta regulation in vertebrates, (C) 2001 Academic Press.