Fig. 4

The tjp3/zo-3 morpholino effect is specific. Analysis of tjp3/zo-3 morpholino injected embryos at 2 dpf showed that the severity of the phenotype is dosage dependent and that the injection of splice-site morpholino and translation morpholino cause the same distribution of phenotypes. (A) Zebrafish embryos were injected at the 1-cell stage with 6 ng/1.5 nl control morpholino, 1.5 nl tjp3/zo-3 splice morpholino (MO) at different concentrations (2 ng, 6 ng, 12 ng) and 1 ng/1.5 nl tjp3/zo-3 translation blocking morpholino (ATGmo). At 2 dpf the phenotypes were assessed by morphological criteria. The death rate was between 20% (2 ng MO) and 40% (12 ng MO). The phenotypes were classified into 4 groups according to severity: no circulation (with edema and curved tail), edema (with or without curved tail), curved tail only, and reduced tailfin only. At a high dose of tjp3/zo-3 morpholino (12 ng) the major phenotype of the survivors was absent circulation with edema and a curved tail (55%), whereas at a low dose (2 ng) it was a reduction of the tailfin (20%) with less than 3% of edema. The injection of only 1 ng of translation blocking morpholino (ATGmo) resulted in a similar distribution of phenotypes as that of 12 ng splice morpholino (MO). Mean and S.E.M.; n ≥ 100 per experiment; 3–5 independent experiments. 2-way ANOVA; p < 0.05 (12 ng MO), p < 0.01 (1 ng ATGmo). Asterisks indicate statistical significance. (B) Phenotypes of injected embryos at 2 dpf. Control morpholino injected embryo (a); tjp3/zo-3 splice morpholino injected embryos (b, d); tjp3/zo-3 translation blocking morpholino injected embryos (c, e). Mild phenotypes presented with crooked tail buds and reduced tailfin size (b, c, arrowheads), while severely affected embryos had no circulation and often an accumulation of red blood cells (d, e, arrows).