The nieuwkoid/dharma Homeobox Gene Is Essential for bmp2b Repression in the Zebrafish Pregastrula

Abstract

Dorsoventral specification of the zebrafish gastrula is governed by the functions of the dorsal shield, a region of the embryo functionally analogous to the amphibian Spemann organizer. We report that the bozozok locus encodes the transcription factor nieuwkoid/dharma, a homeobox gene with non-cell-autonomous organizer-inducing activity. The nieuwkoid/dharma gene is expressed prior to the onset of gastrulation in a restricted region of an extraembryonic tissue, the yolk syncytial layer, that directly underlies the presumptive organizer cells. A single base-pair substitution in the nieuwkoid/dharma gene results in a premature stop codon in boz(m168) mutants, leading to the generation of a truncated protein product which lacks the homeodomain and fails to induce a functional organizer in misexpression assays. Embryos homozygous for the boz(m168) mutation exhibit impaired dorsal shield specification often leading to the loss of shield derivatives, such as prechordal plate in the anterior and notochord in the posterior, along the entire anteroposterior axis. Furthermore, boz homozygotes feature a loss of neural fates anterior to the midbrain/hindbrain boundary. Characterization of homozygous mutant embryos using molecular markers indicates that the boz ventralized phenotype may be due, in part, to the derepression of a secreted antagonizer of dorsal fates, zbmp2b, on the dorsal side of the embryo prior to the onset of gastrulation. Furthermore, ectopic expression of nieuwkoid/dharma RNA is sufficient to lead to the down regulation of zbmp2b expression in the pregastrula. Based on these results, we propose that gastrula organizer specification requires the Nieuwkoop center-like activity mediated by the nieuwkoid/dharma/bozozok homeobox gene and that this activity reveals the role of a much earlier than previously suspected inhibition of ventral determinants prior to dorsal shield formation.

Zebrafish admp is required to restrict the size of the organizer and to promote posterior and ventral development

“…Here, we report the isolation and characterization of a zebrafish admp homologue. We show that its expression is posi-tively regulated by the Bmp repressor Boz (Koos and Ho, 1999;Ryu et al, 2001), but that Admp itself has effects opposite to those of Boz. Thus, the phenotype of boz mutants can be phenocopied by overexpression of admp, whereas boz overexpression phenocopies traits of admp morphants.…”

“…Studies in Xenopus and zebrafish have demonstrated that Bmps act as morphogens inducing ventral cell fates in a dosedependent manner (Harland and Gerhart, 1997;Dosch et al, 1997). On the dorsal side, bmp expression is repressed by the homeodomain protein Bozozok (Boz, also called Dharma or Nieuwkoid; Koos and Ho, 1999;Fekany-Lee et al, 2000). In addition, organizer cells secrete Bmp inhibitors such as Chordin, Noggin, and Follistatin (Mullins, 1999).…”

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.…”

Functional and hierarchical interactions among zebrafish vox/vent homeobox genes

“…␤-catenin cooperates with the HMG-box factor Tcf/Lef in activating the expression of some dorsal-specific genes, such as bozozok/dharma (boz) in zebrafish. Boz, directly regulated by ␤-catenin, is a transcriptional repressor, and its downstream targets include several genes that induce ventral specification or inhibit dorsal development (Yamanaka et al, 1998;Fekany et al, 1999;Koos and Ho, 1999). The zebrafish boz mutant shows defects in dorsoanterior embryonic structures and it has been reported that this factor promotes development of anterior neuroectoderm by inhibiting BMP and Wnt signaling (Solnica-Krezel and Driever, 2001).…”