bozozokdirectly repressesbmp2btranscription and mediates the earliest dorsoventral asymmetry ofbmp2bexpression in zebrafish

Abstract

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 β-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 highaffinity 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.

TGF-β Family Signaling in Neural and Neuronal Differentiation, Development, and Function

“…BMP and Wnt signaling converge later in development when both signaling pathways are integrated through Smad1 phosphorylation of glycogen synthase kinase 3 (GSK3) in gastrulation (Fuentealba et al 2007). In zebrafish, b-catenin induces a homeobox gene bozozok/dharma that directly represses the expression of BMP2b, which encodes BMP-4 (Leung et al 2003). Bozozok 2/2 ; Chordin 2/2 zebrafish mutants show synergistic losses of neural tissue and dorsal structures (Gonzalez et al 2000).…”

Regulation of the early expression patterns of the zebrafish Dishevelled-interacting proteins Dapper1 and Dapper2

“…Boz inhibits the ventralization of zebrafish by directly binding the bmp2b promoter and repressing its expression and by repressing the expression of vox/vega1, vent/vega2, and ved through an undetermined mechanism (Fekany et al, 1999;Koos and Ho, 1999;Kawahara et al, 2000a, b;Melby et al, 2000;Shimizu et al, 2002;Leung et al, 2003a). Boz can also induce ectopic expression of dpr1 and dpr2 (see above), but their expression does not require Boz signaling, as 100% of embryos from boz heterozygous crosses had wild-type dpr1 and dpr2 expression (n ϭ 133 for dpr1 and n ϭ 140 for dpr2; Fig.…”

“…Organizer genes promote dorsal axis formation in three important ways: by antagonizing the activity of ventralizing genes, by establishing dorsal mesendoderm, and by promoting proper gastrulation movements. The direct ␤-catenin target gene bozozok (boz) antagonizes ventral cell fates by repressing ventralizing genes, including bmp2b, vox/ vega1, vent/vega2, ved, and wnt8 (Fekany et al, 1999;Fekany-Lee et al, 2000;Kawahara et al, 2000a, b;Melby et al, 2000;Shimizu et al, 2002;Leung et al, 2003a). Members of the Nodal family, squint (sqt) and cyclops (cyc), are also either direct or indirect targets of ␤-catenin and are responsible for specifying dorsal mesendoderm (Schier et al, 1997;Dougan et al, 2003).…”

Functional and hierarchical interactions among zebrafish vox/vent homeobox genes

“…In the gastrulating embryo, the presence of ved mRNA in the ventral region is the first indication of the Ved role in dorsoventral patterning. Some dorsal factors, such as Gsc, might be responsible for the repression of ved, as well as vox and vent transcription in the dorsal cells (Shimizu et al, 2002;Leung et al, 2003). Furthermore, starting from 50% to 90% epiboly, ved expression is progressively restricted to the marginal zone, in the ventral as well as in the lateral region.…”