Fig. 4. Behaviour of the actomyosin cytoskeleton in th⁵ embryos. (A-D) Actin-phalloidin staining of WT and th⁵ mutant embryos. (A) In WT embryos, actin is found at the apical cell cortex, as well as in small discrete dots. In th⁵ embryos undergoing cell rounding up (B), a subset of the cells show an increased staining with a fibrous appearance. Shortly after cells have rounded up, some cells (right-side of C) maintain a wild-type-looking cortical staining associated with the presence of small dots. The only difference is the change from a columnar cell shape to a rounded cell shape. In the same embryo (left-side of C), some cells show a dramatic delocalisation of actin, with a very faint cortical staining, and dense accumulation of actin in one dense spot at the cortex. In older embryos (D), all cells have delocalised their actin. (E) Embryos lacking DIAP1 were collected 1 hour after cellularisation and extracts were analysed by western blotting using an antibody against the phosphorylated form of Myosin light chain (three independent extracts of 40 embryos each were loaded for each genotype). The blot was reprobed with an antibody against tubulin as a loading control (boxed bands). Band intensities were quantified and the ratio of phospho-MLC to tubulin is indicated in the graph. Shaded boxes show average values across the three samples for each genotype, whereas dotted boxes show the minimum and maximum values found for each genotype. (F,G) Stills from time-lapse supplementary material Movies 8 and 9 of WT and th⁵ mutant embryos labelled with SqhGFP. (F) Wild-type embryos undergoing germ-band extension exhibit a planar polarisation of SqhGFP, with cables running parallel to the dorsoventral (D/V) axis (C+20-50 minutes). In th⁵ embryos (G), SqhGFP planar polarisation is initially normal (C+20 minutes), and then an enrichment of Myosin II is seen in the cables compared with wild-type embryos (C+33 minutes). This enrichment culminates when the embryo contracts, and large regions of the embryo show a strong accumulation of Myosin II in cables as well as in fibrous aggregates at the cell cortices (C+38 minutes). When cells have rounded up (C+50 minutes), Myosin II is found in small dots that are likely to correspond to the membraneous blebs observed in the srcGFP movie (supplementary material Movie 3). Larger dots are also seen, which are likely to be remnants of the basal actomyosin II rings present in wild-type embryos between ectodermal cells and the yolk cell.