Fig. 4

Brd2a protein is reduced in segmentation stage morphants, while maternal protein may persist in early embryos. Levels of Brd2a protein were assessed in uninjected (WT) and 8 ng brd2aMO1-injected (brd2aMO) embryos by immunohistochemistry (A–F wholemounts; I–L sections) and western blot (G, H) using the peptide antibody raised against zebrafish Brd2a. Morphant embryos at prim 5 (A,B) and 18 hpf (B,D) show reduced levels of Brd2a compared to wildtype uninjected embryos (A,C), while early embryos at 4 hpf show equivalent levels (E, F; animal pole view). Western blot shows a major protein band near 75 kD for Brd2a in prim 5 embryos (G), ovaries (H; lane 1, whole ovary; lane 2, stage I-II oocytes), and 0–2 hpf embryos (H; lane 3). Minor bands > 80 kD are often present in ovaries and early embryos, and sometimes prim 5. Putative Brd2a degradation products (or isoforms) near 55 and 37 kD are present in most preparations of prim 5 embryos (G). Reduced levels of Brd2a in brd2aMO1 prim 5 embryos are seen compared to wildtype uninjected controls (G: WT vs. brd2aMO), consistent with immunohistochemical data. Blot was reprobed for β-tubulin as loading control. Brd2a protein is detected in both unfertilized oocytes and 0–2 hpf stage embryos (H; sectioned tissue in J–L), and becomes localized to the cortical periphery of stage III-IV oocytes, tracking with its cognate mRNA (J–L). Negative control IgG in place of anti-Brd2a shows only background staining in tissue sections (I).