|
Fig. 3 Hwa is localized on the plasma membrane of dorsal blastomeres. (A) Illustration of zebrafish Hwa protein structure. Positions of residues at the start of the domain are indicated except the last residue position. (B) Detection of exogenous Hwa-HA by immunostaining using anti-Hwa antibody: 50 pg of hwa-HA mRNA was injected into one blastomere of eight-cell stage Mhwatsu01sm mutant embryos, and the embryos were collected at 4 hpf for immunostaining with anti-HA and anti-Hwa antibodies. Nuclei were also stained with 4′,6-diamidino-2-phenylindole (DAPI). Embryos were imaged by confocal microscopy in animal pole view. Note that exogenous Hwa-HA is enriched on the plasma membrane. Scale bar, 50 μm. (C) Location of endogenous Hwa protein. Zebrafish wild-type and Mhwatsu01sm mutant embryos were collected at the 512-cell stage for coimmunostaining of endogenous Hwa and β-catenin. Embryos were imaged by confocal microscopy and are shown in lateral view with the animal pole to the top. In all 12 wild-type embryos with β-catenin in nuclei of the dorsal blastomeres, endogenous Hwa protein is enriched on the plasma membrane in the same region (top panel). In Mhwa mutants (bottom panel), Hwa signal is absent on the plasma membrane and no blastomeres have nuclear β-catenin. For each embryo, two numbered areas are enlarged for better view. The ratio of embryos with the representative pattern is indicated. Scale bars, 50 μm.