The nieuwkoid/dharma homeobox gene is essential for bmp2b repression in the zebrafish pregastrula

Dorsoventral specification of the zebrafish gastrula is governed by the fun ctions of the dorsal shield, a region of the embryo functionally analogous to the amphibian Spemann organizer. We report that the bozozok locus encode s the transcription factor nieuwkoid/dharma, a homeobox gene with non-cell- autonomous organizer-inducing activity. The nieuwkoid/dharma gene is expres sed prior to the onset of gastrulation in a restricted region of an extraem bryonic tissue, the yolk syncytial layer, that directly underlies the presu mptive organizer cells. A single base-pair substitution in the nieuwkoid/ d harma gene results in a premature stop codon in boz(m168) mutants, leading to the generation of a truncated protein product which lacks the homeodomai n and fails to induce a functional organizer in misexpression assays. Embry os homozygous for the boz(m168) mutation exhibit impaired dorsal shield spe cification often leading to the loss of shield derivatives, such as prechor dal 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 hom ozygous mutant embryos using molecular markers indicates that the boz ventr alized phenotype may be due, in part, to the derepression of a secreted ant agonizer of dorsal fates, zbmp2b, on the dorsal side of the embryo prior to the onset of gastrulation. furthermore, ectopic expression of nieuwkoid/dh arma RNA is sufficient to lead to the down regulation of zbmp2b expression in the pregastrula. Based on these results, we propose that gastrula organi zer specification requires the Nieuwkoop center-like activity mediated by t he nieuwkoid/dharma/bozozok homeobox gene and that this activity reveals th e role of a much earlier than previously suspected inhibition of ventral de terminants prior to dorsal shield formation. (C) 1999 Academic Press.