The vov mutant gene encodes Psmd6. (A) Nucleotide substitution from G to A occurs in a splicing acceptor site of the intron6/7-exon7 boundary (top, underlined), causing a one-base deletion in the psmd6 mRNA that results in a frameshift within the coding region (middle panel) and loss of the PCI domain (bottom panel). (B,C) Ina (B) and Inf (C) of wild type, vov mutant, vov mutant injected with GFP-tagged psmd6 RNA carrying the vov mutation, vov mutant injected with wild-type GFP-tagged psmd6 RNA, psmd6 and psmc2 morphants and their five-mismatch morphants. Ina is lower and the maximum value of Inf is higher in the vov mutant injected with wild-type GFP-tagged-psmd6 RNA (green) than in the vov mutant injected with GFP-tagged-psmd6 RNA carrying the vov mutation (yellow), suggesting that wild-type psmd6 RNA injection rescues the vov-mediated defects in nuclear position and shape of lens fiber cells. Ina is higher and the maximum value of Inf is lower in psmd6 (purple) and pmsc2 morphants (pink) than in their five-mismatch morphants (brown and light blue), suggesting that these MOs induce the vov-like lens defects. (D-I) Expression of zebrafish psmd6 mRNA at the eight-cell stage (D) and at 20 (E), 48 (F,G) and 72 hpf (H,I). (G,I) Plastic sections of eyes. Asterisk indicates psmd6 expression in the retinal CMZ. (J) Semi-quantitative PCR of wild type and vov mutant at 72 hpf using psmd6 and β-actin gene primers. The number of PCR cycles is indicated above each lanes. (K-M) Western blotting of 72 hpf wild type, vov mutant, psmd6 and psmc2 morphants and their five-mismatch morphants with anti-zebrafish Psmd6 (K), anti-polyubiquitylated protein (FK2) (L) and anti-acetylated α-tubulin (M, a loading control). The level of Psmd6 (arrow) is reduced in the vov mutant and psmd6 morphant, but is unaffected in the psmc2 morphant (K). The levels of polyubiquitylated proteins increase in the vov mutant, psmd6 and psmc2 morphant (K). Scale bars: 250 µm in D-F,H; 50 µm in G,I.
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