Figure 1. The mitfa^vc7 intron 6 mutation causes defective temperature-dependent splicing of mitfa.

A. Images of mitfa^vc7 zebrafish embryos (2 days post fertilization) at 24°C (permissive temperature) and 32 °C (restrictive temperature). Pigmented melanocytes are clearly visible on the body of the zebrafish.

B. RT-PCR mitfa RNA expression at 24°C, 26°C, 28.5°C, and 32OC in wild type and mitfa^vc7 mutant embryos. Four types of mitfa transcripts (wt, a, b, c) were consistently observed in mitfa^vc7 embryo across all temperatures while only one transcript (wt) was observed in wild type embryo.

C. Schematic overview of mitfa^vc7 splice variants transcripts and their predicated MITF protein products.

D. Images and quantitation of zebrafish embryos following injection with transgenes expressing the mitfa^vc7 splice variants from the mitfa promoter at 24°C and 32°C. At 24OC, the number of melanocytes promoted by expression of the mitfa⁺ⁱⁿ⁶ splice variant (2.97 (2.27 to 3.67) [mean (95% CI)]) was significantly less active compared to wild type mitfa (8.71 (6.66 to 10.76) [mean (95% CI)]) (p=0.007; ANOVA). In addition, the number of melanocytes promoted by expression of mitfa+in5,6 (1.138 (0.64 to 1.636) [mean (95% CI)]) was significantly less than mitfa (p=0.001; ANOVA) and compared to mitfa⁺ⁱⁿ⁶ (p=0.045; ANOVA).