Bozozok directly represses bmp2b transcription and mediates the earliest dorsoventral asymmetry of bmp2b expression in zebrafish

Formation of the gastrula organizer requires suppression of ventralizing signals and, in fish and frog, the need to counteract the effect of ubiquitously present maternal factors that activate the expression of Bmps. How the balance between dorsalizing and ventralizing factors is shifted towards organizer establishment at late blastula stages is not well understood. Mutations in zebrafish bozozok (boz) cause severe defects in axial mesoderm and anterior neurectoderm and affect organizer formation. The boz gene encodes the homeodomain protein Bozozok/ Dharma and its expression in the region of the organizer is activated through P-catenin signaling. Here, we investigate the molecular mechanism by which boz contributes to the establishment of the organizer. We demonstrate that the homeodomain protein Boz acts as a transcriptional repressor in zebrafish: overexpression of an En-Boz fusion protein can rescue the boz phenotype, whereas a VP16-Boz fusion protein acts as an antimorph. Expression analysis of bmp2b indicates that Boz negatively regulates bmp2b in the prospective organizer. We demonstrate that this Boz activity is independent of that of other zygotic genes, because it also occurs when translation of zygotic genes is suppressed by cycloheximide (CHX). We identify two high-affinity binding sites for Boz within the first intron of the bmp2b gene. Deletion of these control elements abolishes Boz-dependent repression of bmp2b in the early blastula. Thus, Boz directly represses bmp2b by binding to control elements in the bmp2b locus. We propose that early transcriptional repression of bmp2b by Boz is one of the first steps toward formation of a stable organizer, whereas the later-acting Bmp antagonists (e.g. Chordin, Noggin) modulate Bmp activity in the gastrula to induce patterning along the dorsoventral axis. Thus, similar to Drosophila Dpp, asymmetry of Bmp expression in zebrafish is initiated at the transcriptional level, and the shape of the gradient and its function as a morphogen are later modulated by post-transcriptional mechanisms.