Direct binding of Lef1 to sites in the boz promoter may mediate pre-midblastula-transition activation of boz expression.

Abstract

The Nieuwkoop center provides signals essential for the establishment of the dorsal gastrula organizer in vertebrates. Activation of beta-catenin is one of the events in the Nieuwkoop center that lead to activation of dorsal-specific genes during blastula and early gastrula stages. Zebrafish bozozok (boz) mutant embryos have severe defects in axial mesoderm and anterior neuroectoderm. The boz gene is activated in the organizer in response to beta-catenin signaling, and Boz protein has been demonstrated to contribute to organizer formation by repression of ventralizing genes, including bmp2b, vega1, and vega2. Here, we investigate the timing and molecular mechanism by which boz expression is activated in the organizer. We demonstrate that boz is already expressed before midblastula transition (MBT). We further identify high-affinity binding sites for Tcf/Lef1 within the boz promoter region. These sites, together with the finding that beta-catenin induces boz expression, indicate that transcription of boz may be activated directly by beta-catenin/Lef1. We hypothesize that pre-MBT activation of boz may be important to build up a sufficiently strong antagonizing activity against zygotic ventralizing genes activated immediately post-MBT. Thus, the early onset of boz expression may be crucial for organizer establishment in the presence of ubiquitous maternal activators of ventralizing genes.