Figure 7.

mH2A2 and mH2A1 are required for proper zebrafish embryo development. (A) An inhibitory morpholino (MO) was designed against the translational start site of zebrafish mH2A1 or mH2A2 mRNA (sequences at Supplementary Table S12). For the control mismatch morpholino we introduced a few base alterations. Total nuclei of zebrafish embryos were analysed by anti-mH2A1 or anti-mH2A2, and anti-histone H3 immunoblotting after specific morpholino injection. Full-length blots are presented in Supplementary Fig. S11. (B) Characterization of mH2A1 morphants and control embryos at the 18-somite stage. Lateral, apical and dorsal view of fixed zebrafish embryos show alterations in midbrain structure and organization (arrow). Somite number was correct, but their distribution along the embryo was shortened. Yolk sac extension and caudal fin was not properly formed after mH2A1 downregulation. In all experiments we co-injected with a morpholino against p53 (p53 MO) to mitigate the nonspecific dose-dependent neural toxicity widely reported⁵⁶. Rescues were generated by injection of embryos with mH2A1 morpholino in combination with mH2A1.1 mRNA. (C) Characterization of mH2A2 morphants and control embryos at the 10-somite stage. Lateral, apical and dorsal view of fixed zebrafish embryos show alterations in midbrain, optic primordium and somites and fin formation/structure after mH2A2 downregulation. In all experiments we co-injected with a morpholino against p53 (p53 MO) to mitigate the nonspecific dose-dependent neural toxicity widely reported⁵⁶. Rescues were generated by injection of embryos with mH2A2 morpholino in combination with mH2A2 mRNA. (D) Stacked bar graph representing the percentage of embryos which are normal, dead or showing described altered phenotype after injecting control and mH2A1 and mH2A2 morpholinos, and rescued with mH2A1.1 or mH2A2 mRNA at the developmental stages shown in figure (C), where n represents numbers of embryos injected.