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Fig. 6 Ruffle formation in marginal EVL cells is partly restored upon uniform re-introduction of epam via RNA injection, whereas epcam overexpression in single EVL cells is insufficient.
(A–F) Phalloidin stainings of wild type (WT) and maternal zygotic EpCAM mutant (MZ-/-) EVL cells at two different positions, equatorial (A–C) and marginal (D–F); merged Z-stacks of confocal images. In WT embryos, marginal EVL cells are much more protrusive than equatorial EVL cells, while in MZ-/- embryos, EVL cells show practically no protrusive activity at both locations. First four columns of panel (G) show quantification of protrusive activity. (H,I) Phalloidin stainings of marginal EVL cells from un-injected maternal/zygotic EpCAM mutant (MZ-/-) (H) and MZ-/- mutant injected with zebrafish epcam mRNA (I), 80% epiboly stage. There was a partial restoration of the protrusive activity of the EVL cells (red arrow in H), as quantified in (G), columns 5 and 6. Injection of higher amounts of epcam mRNA caused progressive death of injected embryos during pre- or early gastrula stages (data not shown), most likely due to ectopic effects of applied EpCAM in the deep cells, which precluded analyses of the EVL phenotype. (J) Injection of plasmid DNA encoding EpCAM-GFP fusion protein under the control of CMV promoter in MZ-/- embryos led to high recombinant protein levels in single cells. In most cases, this also caused death of expressing cells (data not shown). The few surviving EVL cells failed to restore ruffles (n = 0/13), suggesting that ruffle formation also requires EpCAM function in adjacent EVL cells, to which the protrusions usually attach (compare with Figure 7). Unfortunately, in over 100 investigated embryos, we failed to obtain clones with adjacent EpCAM-GFP-positive EVL cells, as would have been necessary to directly test this notion.