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Fig. 2. Delay in metaphase-anaphase transition requires prior irradiation at specific parts of the cell cycle. (A) Metaphase index, the ratio of cells in metaphase to those in anaphase and telophase (M/A+T), is maximal at 40 minutes after irradiation. Wild-type embryos in cycle 16 (330-390 minutes AED for non-irradiated controls and 310-370 minutes AED for irradiated embryos) were treated with 8.3 Gy of X-rays (LD₈₀ dose) and fixed 40, 60 or 90 minutes later. Embryos were stained with an antibody to phosphorylated histone H3 (PH3), a mitotic marker, and Hoechst33258 to visualize DNA. The number of metaphases, anaphases and telophases within the dorsal ectoderm was quantified and the metaphase index calculated. Each bar represents approximately 1000 cells from at least 10 embryos. (B) Irradiation with an LD₈₀ dose during S phase, but not G₂/M, delayed metaphase-anaphase transition. Schematic of the 15th-16th mitotic cell divisions within the lateral ectoderm. The approximately 60 minutes mitotic cell cycle is separated into M phase (10 minutes), followed directly by S (35-45 minutes) and G₂ phase (10 minutes). Numbers 1 and 2 mark the approximate place within the cell cycle in which cells were irradiated (see below). (C) The ability to delay metaphase-anaphase transition is dependent upon the dose of irradiation and cell cycle phase. Wild-type embryos with ventral ectoderm in cycle 15 and dorsal ectoderm in cycle 16 (330-360 minutes AED) were irradiated with 0, 8.3 Gy (LD₈₀) or 83 Gy (10xLD₈₀) of X-rays. Those irradiated with LD₈₀ were allowed to recover for 40 minutes before analysis. Therefore, based on embryonic age, mitotic cells analyzed were in S phase at the time of irradiation (point 1 in B). For irradiation in G₂/M, embryos were irradiated and immediately analyzed without an intervening recovery period (point 2 in B approximately 2-10 minutes after IR). Each bar represents at least eight nuclei from six embryos. (D) The mei-41^D12 mutants can regulate the metaphase-anaphase transition after exposure to 10xLD₈₀. Wild-type and mei-41^D12 mutant embryos were irradiated with a LD₈₀ dose and allowed to recover for 40 minutes before analysis (data reproduced from Fig. 1C for direct comparison), or irradiated with a 10xLD₈₀ dose and analyzed immediately. 10xLD₈₀ sample includes mitotic cells in the ventral ectoderm (cycle 15) and the dorsal ectoderm (cycle 16). For 10xLD₈₀, at least eight nuclei from seven different embryos were analyzed. As in Fig. 1, asterisks denote statistically significant data with P<0.0001. All values were compared with non-irradiated values.

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