Figure 7.

Functional assay of zebrafish cdc6^tsu21cd and human CDC6(T323R) alleles in zebrafish cdc6^tsu4305 mutant embryos. (A) Illustration of zebrafish and human mutant forms of Cdc6. The D-box, KEN-box, Cy-motif, ATPase domain, DBD, phosphorylatable serines (*) were indicated. Morphology (B) and TUNEL assay results (C) of cdc6^tsu4305 mutant embryos at 24 hpf. Embryos derived from heterozygous fish crosses were injected with indicated mRNA at the one-cell stage, and analyzed at 24 hpf, which was followed by genotyping of individuals. The ratio of embryos with representative morphology or signals was shown in the right corner of each picture. In (C), the boxed areas of several TUNEL confocal pictures were enlarged for easier comparison. (D) Up-regulated expression of tp53 and bbc3 in cdc6^tsu4305 mutants at 22 and 24 hpf as analyzed by RT-PCR. WT, wild-type siblings; MT, mutants. (E) Relative expression levels of tp53 and bbc3 in cdc6^tsu4305 mutants at 24 hpf. Embryos injected with indicated mRNA (100 pg per embryo) were individually lysed at 24 hpf. After genotyping using an aliquot of cells, the remaining cells of embryos homozygous for cdc6^tsu4305 allele were mixed and the isolated RNA was used for RT-PCR. In (D, E), statistical significance levels are: **, P < 0.01; ***P < 0.001.