Fig. 3. The role of grapes in regulation of mitosis after DNA damage. (A) grp¹ mutants delay entry into mitosis after irradiation. Wild-type (Wt) and grp¹ embryos in cycle 16 (325-335 minutes AED) were irradiated with 0 or 8.3 Gy of X-rays and fixed 20 minutes later to visualize the delay in entry into M16. Embryos were stained with an antibody to PH3, Hoechst33258 to visualize DNA, and an antibody to ß-gal to identify homozygous mutants. At the time of fixing, cells of the dorsal ectoderm (enclosed with brackets) enter M16 in untreated embryos (`Wt-IR' and `grp¹–IR'), but were delayed in the entry in irradiated embryos (`Wt+IR' and `grp¹ +IR'). Embryos are shown with anterior to the left and ventral side down. (B) Quantification of data in A. Mitotic index decreased following irradiation in both wild type and grp¹ embryos, indicating that both genotypes could regulate the entry into mitosis in response to damaged DNA. Mitotic index was quantified within the dorsal ectoderm for at least 10 embryos per genotype. (C,D) grp¹ mutants show an increase in metaphase index after irradiation. Wild type and grp¹ and mutant embryos in cycle 16 (330-360 minutes AED for non-irradiated controls and 310-370 minutes AED for irradiated embryos) were irradiated with 0 or 8.3 Gy of X-rays and fixed 0 (–IR) or 40 minutes (+IR) later. Embryos homozygous for a deficiency (Df/Df) that removes grapes [Df(2)H20] also showed a robust increase in metaphase index after irradiation. Metaphase index in the dorsal ectoderm was quantified as in Fig. 2 for at least 29 embryos per treatment for each genotype (D). PH3 staining of a representative sample is shown in C where arrowheads mark anaphase and telophase nuclei and arrows mark metaphase nuclei. (E) Live analysis confirmed that grp¹ mutants delay metaphase-anaphase transition after irradiation as indicated by analysis of fixed embryos in A-D. Wild type and grp¹ embryos in cycle 16 (330-360 minutes AED) were irradiated with 0 or 8.3 Gy of X-rays and analyzed 0 (–IR) or 40 minutes (+IR) later. Each bar represents at least 10 nuclei from four different embryos. The data for mei-41^D12 mutants is reproduced from Fig. 1 for comparison. As in Fig. 1, asterisks denote statistically significant data with P<0.0001. All values were compared with non-irradiated values.