JCS03472 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Adobe PDF

• Supplemental Figure S1 -

  Fig. S1. Cell divisions cease in th⁵ embryos following cell rounding and dispersal. Staged collections were analysed for patterns of active cell division using an antibody to phosphorylated Histone H3. WT embryos show dynamic patterns of cell division occurring in specific domains at stage 8 (A) and 9 (B). B’ shows a higher magnification of the boxed region in B. The mitotic figures show cells in various stages of division from prophase to telophase. Prior to cell rounding and dispersal, the pattern of division in th⁵ mutant embryos is identical to WT (C). Soon after cell rounding, however, cell division ceases in th⁵ mutants (D), and only a few cells are still positive for Histone H3 staining (D’). These are presumably the nuclei of cells that had already entered mitosis before the onset of apoptosis, and could not complete division because the nuclei started condensing. As a consequence, the chromatin has condensed as a dot in these nuclei.

• Supplemental Figure S2 -

  Fig. S2. Blocking cell death by overexpressing p35 in th⁵ embryos rescues cell dispersal. The maternal driver tubulinGal4VP16 was used to overexpress p35 ubiquitously in th⁵ embryos. Embryos were fixed 1 hour after cellularisation and double-stained for (1) Engrailed to assess cell dispersal and (2) TUNEL to monitor cell death. In WT embryos stained for En (A), no cell death is detectable by TUNEL (B). At the same time-point, th⁵ embryos show rounded and dispersed cells (C), which are all positive for TUNEL (D). By contrast, in th⁵ embryos expressing p35, 92% (n=84) of th⁵ homozygous mutant embryos showed a rescued phenotype, with rescue of the cell dispersal (E) and rescue of cell death (F). Note that because of the variability of Gal4VP16 loading in embryos (see Materials and methods), the extent of rescue is variable with 55% (n=84) of embryos showing a complete rescue, i.e. a phenotype indistinguishable from WT embryos, and 37% (n=84) showing an incomplete rescue, where cell death was rescued but the morphology of the embryo was not completely normal (as in the embryo shown in E and F).

• Supplemental Figure S3 -

  Fig. S3. Overexpression of Reaper, but not Ricin, results in cell dispersal and cell death. The maternal driver tubulinGal4VP16 was used to overexpress Reaper (Reaper^OVER) and Ricin (Ricin^OVER) ubiquitously in embryos. Reaper overexpression leads to cell dispersal (shown by Engrailed staining, A) and caspase activation (shown by staining of activated Drice, B). By contrast, overexpression of Ricin results in inhibition of translation, evident by the loss of En expression, but cells do not appear to disperse (C). The absence of cell dispersal was confirmed by labelling the Engrailed-expressing cells using in situ hybridisation to detect Engrailed mRNAs (D).

• Supplemental Figure S4 -

  Fig. S4. Inhibition of Myosin light chain activity by injection of Y27632 in th⁵ embryos. Pre-cellularised embryos were injected with either the Drok kinase inhibitor Y27632 or H₂0 as a control, then fixed and stained for Engrailed. Wild-type embryos injected with Y27632 fail to undergo germ-band extension (A), whereas control embryos injected with H₂O develop normally (B), showing that Y27632 effectively disrupts Myosin II activity through Drok kinase inhibition (see also Bertet et al., 2004). When th⁵ embryos are injected with Y27632 (C), a partial rescue of the th⁵ phenotype is observed, in contrast to control th⁵ embryos injected with H₂0 (D). The yolk cell (y) does not fragment and there is a reduction in the extent of the cell multi-layering (compare also with Reaper overexpression, Fig. 3B and Fig. 3F, Movie 6).

• Movie 1 -

  Movie 1. Confocal movie of th⁵ homozygous embryo: nuclei are labelled with HistoneYFP. Genotype: UASH2BmYFP/+; tubulinGal4VP16.th⁵/th⁵, abbreviated as HistoneYFPth⁵.

• Movie 2 -

  Movie 2. Nomarski movie of th⁵ homozygous embryo.

• Movie 3 -

  Movie 3. Confocal movie of th⁵ homozygous embryo: membranes are labelled with SrcGFP. Genotype: UASsrcGFP/+; tubulinGal4VP16.th⁵/th⁵, abbreviated as SrcGFPth⁵.

• Movie 4 -

  Movie 4. Nomarski movie of embryo overexpressing Reaper. Genotype: tubulinGal4VP16/UASreaper, abbreviated as Reaper^OVER.

• Movie 5 -

  Movie 5. Nomarski movie of embryo overexpressing Ricin. Genotype: tubulinGal4VP16/UASricin, abbreviated as Ricin^OVER.

• Movie 6 -

  Movie 6. Confocal movie of embryo overexpressing Reaper: the adherens junctions are labelled with DECadherinGFP: Genotype: tubulinGal4VP16; DEcadGFP/UASreaper, abbreviated as DEcadGFP Reaper^OVER.

• Movie 7 -

  Movie 7. Confocal movie of embryo overexpressing Ricin: the adherens junctions are labelled with DECadherinGFP. Genotype: tubulinGal4VP16; DEcadGFP/UASricin, abbreviated as DEcadGFP Ricin^OVER.

• Movie 8 -

  Movie 8. Confocal movie of wild type embryo: Myosin II is labelled with SqhGFP. Genotype: SqhGFP42; SqhGFP40, abbreviated as SqhGFP.

• Movie 9 -

  Movie 9. Confocal movie of th⁵ homozygous embryo: Myosin II is labelled with SqhGFP. Genotype: SqhGFP42; SqhGFP40.th⁵/SqhGFP40.th⁵, abbreviated as SqhGFPth⁵.