Fig. 5

Rescue of the phenotype. Tjp3/zo-3 morphants can be rescued by full-length zebrafish tjp3/zo-3 and a truncated zebrafish tjp3/zo-3 without the C-terminal PDZ-binding motive, but not dog TJP3/ZO-3. (A) tjp3/zo-3 morphants can be rescued by co-injection of tjp3/zo-3 splice morpholino and pcDNA3.1 expression vector containing full-length Flag-tagged zebrafish tjp3/zo-3 (zf tjp3) or mutant zebrafish tjp3/zo-3 without the PDZ-binding motif (zf tjp3ΔP). Co-injection of pcDNA3.1 dog TJP3/ZO-3 cannot rescue the morphant phenotype, even tough the protein is expressed and enriched in membrane associated protein fractions of zebrafish (data not shown). Phenotypes were counted at 1 dpf. Mean and S.E.M.; n ≥ 100 per experiment; 2–6 independent experiments. 2-way ANOVA; p < 0.01. Asterisks indicate statistical significance. (B) Rescued embryos showed a straight body axis, little (arrowhead) or no edema, and only mild tailfin malformations (arrow). Non-rescued embryos presented with the morphant phenotype as described in Fig. 2 and Fig. 4. (C) Immunofluorescence on cryosections of WT embryos (a, b) and embryos injected with pcDNA3.1 Flag-tagged zebrafish tjp3/zo-3 (c, d) at 2 dpf. WT embryos were labeled with Tjp3/Zo-3 antibody to detect endogenous Tjp3/Zo-3 localization (a, b). Ectopically expressed Tjp3/Zo-3 localizes accurately. Anti-Flag staining was detected in the apical side of the pronephric ducts (c, arrowheads) and in the skin (d). (D) Anti-Flag Western blot at 2 dpf. Embryos were injected with control morpholino (Cmo), pcDNA3.1 zebrafish tjp3/zo-3 (zf tjp3), or co-injected with morpholino and pcDNA3.1 zebrafish tjp3/zo-3 (rescue). Ectopic Flag-tagged Tjp3/Zo-3 is expressed in embryos injected with tjp3/zo-3 alone (lane 1) as well as in the rescued embryos (lane 2). No Flag labeling was detected in controls (lane 3, different experiment). (E) RT-PCR analysis was performed using total RNA of 2 dpf embryos to check for the activity of the splice morpholino in rescued embryos. In control embryos (lane 1) and in morpholino injected embryos without phenotypes (lane 3), the properly spliced 97 bp wt band was amplified predominantly. However, tjp3/zo-3 morphants had a strong unspliced band at 210 bp (lane 2), which was also present in rescued embryos (lane 4).