Supplementary Figure 6: Effects of inhibitors on development and Myosin light chain phosphorylation.

(a) Left panels phosphorylated Myosin light chain (green) and actin staining (red) for a control embryo incubated for 6 h in-ovo followed by 2 h in EC culture, middle panels, an embryo incubated for 6 hours in-ovo followed by 2 h treatment with 50 μM of the Rho kinase inhibitor H1152 in EC culture, resulting in a partial inhibition of Myosin regulatory light chain phosphorylation, right panels an embryo incubated for 6 hours in-ovo followed by 2 h treatment with 50 μM of the Blebbistatin in EC culture showing only very limited inhibition of Myosin light chain phosphorylation. (b) Upper panel, embryo developed for 16 h in the presence of 50 μM Blebbistatin showing a streak indistinguishable in shape from control embryos. Lower panel an embryo developed in the presence of 50 μM, H1152 showing a characteristic short wide streak. (c) Myosin Ib (green) and actin (red) staining in the posterior epiblast in an embryo incubated for 6 h in ovo, followed by either 2 h in EC culture, or 2 h in EC culture containing 5uM PCP. (c) Myosin Va (green) and actin (red) staining in the posterior epiblast in an embryo incubated for 6 h in ovo, followed by either 2 h in EC culture, or 2 h in EC culture containing 5uM PCP. The results show are representative for several experiments Blebbistatin (control n = 11 embryos/10 streaks/3 experiments, Blebbistatin 10 streaks/12 embryos/3 experiments), HH1152 experiments (control n = 35 streaks/38 embryos/5 experiments, HH1152 n = 28 streaks/33 embryos/5 experiments). White Scale bars in a,c,d, 25 μm. White scale bar in b 1 mm.