|
Fig. 7 Abnormal cell shape in mypt1 mutants and rescue with myosin II inhibitor. (A) Experimental outline for blebbistatin rescue of cell shape in zebrafish mypt1 mutants. (1) Injection of memGFP mRNA into mypt1 mutant clutch. (2) Embryos at 17 hpf treated with 0.1% DMSO or 50 μM blebbistatin. (3) Live imaging of the hindbrain by laser-scanning confocal microscopy. (4) PCR identification of the wild-type and mypt1 mutant embryos imaged. (B) Blebbistatin (bleb) rescue results. (a,e) Wild-type embryo treated with DMSO. Cells were outlined (a) and 3D reconstructed (e). (b,f) Wild-type embryo treated with blebbistatin. Cells were outlined (b) and 3D reconstructed (f). (c,g) mypt1 mutant treated with DMSO. Cells were outlined (c) and 3D reconstructed (g). (d,h) mypt1 mutant treated with blebbistatin. Cells were outlined (d) and 3D reconstructed (h). Images are representative of three independent experiments. At least four embryos for each treatment group were analyzed for cell shape and three to five cells were outlined from each embryo. Yellow, rhombomere cells; blue, boundary cells. Asterisks indicate the ear. Anterior is to the left. (C) Quantitation of neural tube width at the widest region of r4 (or at the boundary between r4 and r5, see Fig. S9 in the supplementary material) in embryos treated with DMSO or blebbistatin as indicated (wild type+DMSO, n=7; wild type+bleb, n=8; mypt1+DMSO, n=6; mypt1+bleb, n=5). *, P<0.01 (Mann-Whitney U-test). Scale bars: 25 μm.