(a and b) The G to A substitution in splicing acceptor site of exon 12 in REP1 genes induced zebrafish evanescence (eva^rk10) mutant having abnormal stop codon by aberrant splicing. (b) eva^rk10 mutant protein lacks the C-terminal GDI domain of the wild-type REP1 protein of zebrafish. (c) Morphological phenotypes of WT and eva^rk10 mutant embryos were examined with dissecting microscope at 5 day post fertilization (dpf). Arrows indicate abnormal morphology of head, pharyngeal arch, swim bladder (black), and eye (red). (d) Apoptosis was visualized by TUNEL assay in WT and eva^rk10 mutant embryos at 5 dpf. (e) Paraffin-embedded zebrafish tissues were subjected to immunohistochemistry using anti-active caspase3 antibody. Eye: red arrow; Tectum: black arrow; Cerebellum: arrowhead Magnification: x50; scale bar = 100 µm. Similar results were observed in two independent experiments.

(a) Zebrafish frozen tissue sections were subjected to immunofluorescence assay using anti-EGFR antibody. Pharyngeal arch: white arrows; Esophagus: green arrow; EGFR: red; Nucleus; blue. Magnification: x50, Scale bar = 100 µm. (b) Paraffin-embedded zebrafish tissues were subjected to immunohistochemistry using anti-EGFR antibody. Scale bar = 100 µm. (c) Cell lysates from WT and eva^rk10 mutant embryos at 5dpf were processed for immunoblot analysis using anti-EGFR and β-actin antibodies. β-actin was used as a loading control. Similar results were observed in two independent experiments.