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Fig. S2 Sequences and specificity of fzr1-MO. (A) Alignment of the target sequences of antisense fzr1-MO and the corresponding sequences of five-mismatch fzr1 mRNA. Mismatched nucleotides are indicated in red. The deduced amino acid sequence of five-mismatch fzr1 mRNA is identical to that of wild-type fzr1 mRNA. The codon of the initial methionine is underlined. (B,C) 72 hpf embryos injected with fzr1-MO. These embryos were classified into two groups showing morphologically severe (B) and mild (C) defects. (D) Pie charts for severities of morphological defects in wild-type embryos injected with distilled water (DW), five-mismatch fzr1 RNA (50 µg/ml), fzr1-MO (0.25 mM) and both MO and mRNA. The injection of wild-type fzr1 RNA (data not shown) or five-mismatch fzr1 RNA into wild-type eggs causes severe malformation and embryonic death during gastrulation, suggesting that a high level of fzr1 RNA is toxic. However, the injection of five-mismatch fzr1 RNA decreases the morphological defects in fzr1 morphants as well as the rate of embryonic death, suggesting a counterbalance between five-mismatch fzr1 RNA and fzr1-MO. (E-F′) Ventral views of the Cecyil transgenic embryos injected with fzr1-MO (E′) and with both fzr1-MO and five-mismatch fzr1 mRNA (F′). (E,F) Green channel (mAG-Gem) only. Injection of fzr1-MO induces the colocalization of mAG-zGem (green) and mKO2-zCdt1 (red) (E,E′), suggesting that APC/C-mediated degradation of mAG-Gem is compromised. By contrast, the injection of both fzr1-MO and five-mismatch fzr1 mRNA inhibits the accumulation of mAG-Gem in lens fiber cells (F,F′), suggesting that five-mismatch fzr1 RNA inhibits defects in the degradation of mAG-Gem in the fzr1 morphant. (G) The percentage of mAG-zGem-positive cells relative to the total number of mKO2-zCdt1-positive lens fiber cells. The fraction of lens fiber cells expressing both mAG-zGem and mKO2-zCdt1 is significantly decreased in the fzr1 morphant injected with five-mismatch fzr1 RNA (P<0.05). The P-value was determined by Student′s t-test using n=9 for the fzr1 morphant and n=10 for the fzr1 morphant injected with five-mismatch fzr1 RNA. Scale bars: 50 µm.