Effects of plakoglobin knockdown on zebrafish development. (A, C, E) Control 5 bp mismatch morpholino or (B, D, F) plakoglobin morpholino, 5 ng, were injected into embryos at 1- to 2-cell stage. Phenotypes were observed at (A and B) 24 hpf, (C and D) 48 hpf, and (E and F) 72 hpf. At 24 hpf, (B) morphant embryos displayed oedema (arrowhead), reduced heart size and a delay in midbrain–hindbrain boundary formation. At 48 hpf, (D) morphant embryos displayed cardiac oedema, reduced heart size, kinked tail, blood pooling (asterisk) compared to (C) controls. At 72 hpf, (F) morphant embryos displayed defects including cardiac oedema, blood pooling and a kinked tail. The numbers of embryos that displayed the above phenotypes were (A) 120/129 (B) 111/137 (C) 120/129 (D) 20/137 (E) 120/129 (F) 24/137. (G) Either 2.5 ng, 5 ng, or 7.5 ng, of plakoglobin morpholino was injected into embryos at 1- to 2-cell stage and the phenotypes scored at 24-, 48-, and 72-hpf. The percentage of embryos classed as normal (blue), mild (maroon), intermediate (yellow), severely affected (light blue) or dead (purple) for each dose at each timepoint are displayed. At 24 hpf, a similar percentage of embryos was affected at all doses. However, by 48 hpf, embryos injected with 2.5 ng appeared normal. 50 embryos were injected per experiment, n = 3 separate experiments. (H–K) Lateral views of 72 hpf plakoglobin morphant embryo hearts with anterior to left and dorsal to top. The cardiac phenotype ranged from a (H) normal phenotype similar to control injected embryos 49.15%, (I) mildly affected 16.62%, (J) intermediate 14.61% and (K) severely affected 19.65% morphant embryos alive at 72 hpf n = 129. (L) Western blot analysis of 72 hpf plakoglobin- or control-morpholino injected embryos, phenol red injected vehicle control and wildtype embryos using anti-plakoglobin antibody. Plakoglobin was detected in control morpholino injected embryos, phenol red and wildtype embryos (arrow) but not in morphant embryos confirming knockdown of the protein by the morpholino. An anti-α-tubulin antibody was used as a loading control.
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