Dorsal axis disruption in brom bones mutant embryos. (A-F) Dorsal gene expression is reduced and ventral gene expression is expanded in brom bones mutant zebrafish embryos, as revealed by whole-mount in situ hybridization. Compared with wild type (A), chordin is variably reduced at 50% epiboly in brom bones mutants (B): 24% showed moderately reduced (B), 24% showed strongly reduced (B′), 25% had absent and 27% had normal (data not shown) chordin expression (n=59). Arrows mark lateral extent of expression. (C-D′) Compared with wild type (C), gata2 showed variable expansion in ventral tissue (denoted by brackets) in brom bones mutants at 50% epiboly: 25% showed partial expansion (D), 39% showed complete expansion (D′) and 36% had normal (data not shown) gata2 expression (n=51). squint expression marks the future dorsal side at midblastula stages in wild-type embryos (E, arrows), but was absent (F, arrows; 29%), severely reduced (38%) or normal (33%, data not shown) in brom bones mutant embryos (n=45). (G,H) β-catenin protein is present in the nuclei (arrows) and at the plasma membrane in wild-type embryos (G), but was only present at the plasma membrane of brom bones mutant embryos (H, 29%, n=52). The remaining mutant embryos showed either a reduced (42%) or normal (29%) domain of nuclear-localized β-catenin (data not shown). (I) Western blot of 1-cell-stage lysates shows that β-catenin protein abundance was not affected in brom bones mutants. β-tubulin is a loading control. d, dorsal; v, ventral.