Cytohesins Downregulate Myosin and Promote Tissue Elongation for Zebrafish Epiboly

(A) Differential interference contrast (DIC) imaging of embryos injected with a cytohesin morpholino cocktail (MOs) are delayed in epiboly (red arrows) compared with uninjected embryos that reach ∼90% epiboly (white arrows) in the same time. Figure S4A shows the expression pattern of the four targeted cytohesin genes.

(B) In situ hybridization of the leading-edge marker ta reveals ∼90% epiboly of uninjected embryos, a delay with the morpholino cocktail (red arrows), a partial rescue with step mRNA co-injection, and failed rescue with step^E173K mRNA co-injection (red arrow). Injection of step mRNA or step^E173K mRNA alone had no effect. The embryos are oriented with the animal pole at top. Quantifications of embryo populations are below.

(C) Embryos with normal or delayed epiboly were stained for F-actin (phalloidin) and imaged by confocal microscopy. Delayed epiboly correlated with smaller apical areas of ectodermal (EVL) cells.

(D) Embryos expressing non-muscle myosin light-chain 12-GFP (myl12.1-GFP) were injected with mismatched or cytohesin morpholinos and imaged live prior to 50% epiboly. The images are Imaris Surpass View maximum-intensity projections. Note the myosin enrichment at tri-cellular junctions in each case, but that the cytohesin morpholino cocktail elicited increased myosin localization to cable-like structures (yellow arrows) connecting the tri-cellular junctions. In each of two experiments, the cytohesin morpholino cocktail resulted in enhanced myosin cables (for n = 8 and 5 embryos in each experiment) not observed with the mismatched morpholinos (for n = 4 embryos in each experiment) or without injection (for n = 2 and 6 embryos in each experiment). Figure S4B shows further mismatched morpholino analysis.

Expression and perturbation of zebrafish cytohesin genes (Related to Figure 5). (A) In situ hybridization detects the expression of four cytohesin genes (1a, 1b, 3a and 4a) in the blastoderm during epiboly. Each was undetectable in the yolk (base of each embryo). These data show that the cytohesins targeted by the morpholino cocktail are normally expressed in the blastoderm, the tissue disrupted by the morpholino cocktail (Figure 5A-D). (B) The cytohesin morpholino cocktail (targeting cytohesins 1a, 1b, 3a and 4a) consistently produced a severe delay in epiboly not observed in embryos injected with the mismatch morpholino cocktail nor in uninjected embryos (seen in three separate experiments; 20-30 embryos were injected for each cocktail in each experiment). Controls for data shown in Figure 5A-D.