DEV02831 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Histone H3 dephosphorylation on mitotic exit. Histone H3 is phosphorylated during mitosis and grp mutations have been reported to accelerate dephosphorylation of this protein during early anaphase (Su et al., 1999). Embryos were double labeled for the phosphorylated form of Histone H3 (PH3) and DNA, and were then analyzed by laser scanning confocal microscopy. In both wild type and in grp mutants, phosphorylated Histone H3 persists through anaphase and declines during telophase. (A-C) Anaphase of mitosis 10 in wild type; (D-F) telophase of mitosis 10 in wild type; (G-I) anaphase of mitosis 10 in grp; (J-L) telophase in mitosis 9 in grp.

• Supplemental Figure 2 -

  Fig. S2. Cyclin A expression in syncytial embryos. Whole-embryo homogenates were subjected to SDS-PAGE and Cyclin A levels were assayed by western blotting. Actin was used as a loading control. (A) In 0-90 minute embryos, Cyclin A is expressed at similar levels in wild types, grp mutants, mnk mutants and mnk grp double mutants. (B) In 0-180 minute embryos, Cyclin A is expressed at higher levels in grp mutants. However, levels are restored to wild type in mnk grp double mutants. Overexpression of Cyclin A in later syncytial blastoderm-stage grp mutants thus appears to be a secondary consequence of Chk2 activation.

• Supplemental Figure 3 -

  Fig. S3. runt expression in cellularization-stage embryos treated with bleomycin. Enhanced fluorescence in situ hybridization was used to assay runt expression in control w¹¹¹⁸ (w) and mnk (mnk) mutant embryos following treatment with bleomycin (+bleomycin) or carrier (−bleomycin). All embryos that had cellularized, regardless of genotype, expressed runt in the typical seven-stripe pattern (A-D). By contrast, w¹¹¹⁸ embryos showing cytological indications of DNA damage, including a disorganized nuclear monolayer, did not express runt at detectable levels (Fig. 5). Scale bar: 100 μm.

• Movie 1 -

  Movie 1. DIC time-lapse recording of a wild-type (w¹¹¹⁸) embryo.

• Movie 2 -

  Movie 2. DIC time-lapse recording of a mnk mutant embryo.

• Movie 3 -

  Movie 3. DIC time-lapse recording of a grp mutant embryo.

• Movie 4 -

  Movie 4. DIC time-lapse recording of a mnk grp double-mutant embryo.

• Movie 5 -

  Movie 5. Time-lapse recording of a wild-type (w¹¹¹⁸) embryo injected with bleomycin and rhodamine-labeled tubulin immediately before the MBT. (Left) DIC recording; (right) rhodamine-tubulin. This embryo never cellularizes.

• Movie 6 -

  Movie 6. Time-lapse recording of a mnk mutant embryo injected with bleomycin and rhodamine-labeled tubulin immediately before the MBT. (Left) DIC recording; (right) rhodamine-tubulin. This embryo cellularizes.