Figure 4.

Activation of Atm-Chk2 pathway in udu^tu24 mutants. (A) Real-time PCR of atm, atr, chk1 and chk2 mRNA levels extracted from 22 hpf embryos. The histogram is depicted as average ± SD format from three independent experiments (p < 0.05, Student's t test). The transcript level of atm and chk2 are significantly up-regulated in udu^tu24 mutants. (B and C) Western analyses showed increased levels of Chk2 and phospho-Chk2 (Ser33) in udu^tu24 embryos compared with wild-type embryos at 22 hpf. (D–H) Neutral single-cell electrophoresis or comet assay of untreated wild-type cells (D), untreated udu^tu24 cells (E), cells from p53-MO–injected udu^tu24 embryos (F), cells from p53-MO–injected wild-type embryos (G), and wild-type cells (H) isolated from embryos irradiated with UV for 1 h. The head is composed of intact DNA, whereas the tail consists of DNA with DSB. (I–K) TUNEL staining of udu^tu24 embryos treated with DMSO (I), 15 μM KU55933 (J), and 200 μM CGK733 (K) at 26 hpf. The number of TUNEL-positive cells is significantly reduced in embryos treated with KU55933 (62.5%; n =5/8) and CGK733 (80%; n = 8/10). (L–N) Phenotypes of chk2 morphants at 21 hpf. (N) Homozygous udu^tu24 embryos injected with chk2-MO have tail-tips that were not round-shaped but tapered (arrowhead). Somite structures are restored in 2.08 pmol chk2-MO–injected udu^tu24 embryos, compared with (M) noninjected udu^tu24 embryos. (O–Q) TUNEL staining of embryos at 21 hpf. (Q) The number of TUNEL-positive cells is significantly reduced in chk2-MO–injected udu^tu24 embryos, compared with (P) noninjected udu^tu24 embryos. (L and O) Wild-type embryos as the positive control for phenotypic observations and TUNEL assay, respectively.