The vclb gene is disrupted in the v12 mutant. (A) The zebrafish vclb gene is a homolog of human vinculin (VCL). (B) vclb mRNA expression as revealed by in situ hybridization is similar to EGFP expression patterns in the somites at 24 hpf and the heart at 48 hpf. Arrow points to the heart. (C) vclb is an alternative transcript corresponding to the vcla transcript variant without exon 19. No other alternative splicing transcripts are detected. odc (ornithine decarboxylase 1) is a loading control. RT–, without reverse transcriptase. (D) Diagram indicating that the transposon insertion in the v12 mutant causes fusion of EGFP with the N-terminal 261 amino acids of Vclb (NVclb). (E) NVclb-EGFP fusion protein expression detected with a GFP antibody in heterozygous and homozygous v12 mutant heart at 30 dpf. Size markers (kDa) are shown to the right. Gapdh provides a loading control. (F,G) Intact vclb, but not vcla, mRNA expression is blocked in the v12 mutant at 36 hpf as assayed by RT-PCR (F) and qRT-PCR (G). The expression level of vcla is the same among wild type, heterozygotes and v12 mutant. The qRT-PCR data are presented as mean±s.e.m. Two-tailed unpaired t-test; NS, not significant; ***P<0.001.
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