Fig. 3. DNA double-strand-break accumulation in grp mutant embryos. (A-F) To assay for DNA damage in grp mutants, embryos were stained with an antibody that recognizes a phosphorylated form of histone H2AX (Drosophila homolog: Histone H2Av) (anti-H2AX), and for DNA with TOTO3. (A-C) H2AX labeling is not observed in early grp mutant embryos. (D-F) However, H2AX labeling becomes prominent in grp mutant embryos after mitosis 13. Cellularization consistently fails in these mutants (Sibon et al., 1997). (C,F) DNA, red; phospho-histone-H2Av, green. (G) Chk2 phosphorylation in grp mutant embryos. Extracts from developmentally staged wild-type (w¹¹¹⁸) and grp mutant embryos were assayed for Chk2 expression by western blotting. Chk2 shows reduced electrophoretic mobility, characteristic of activating phosphorylation, in 3-4 hour grp mutants, but not in wild-type controls. Scale bar: 10 µm.