A-crystallin expression prevents -crystallin insolubility and cataract formation in the zebrafish cloche mutant lens

DEV02424 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Retina photoreceptor. Sections were prepared from 3.5 and 5 dpf embryos, and analyzed by immunohistochemistry with antibodies that detect photoreceptors (green). Photoreceptor and retinal thickness are the same in cloche as in wild type. In addition, the photoreceptor structure in cloche does not appear to change from 3.5 to 5 dpf.

• Supplemental Figure 2 -

  Fig. S2. crystallin insolubility and rescue in cloche^s5. (A) Western blot: total cloche^m39 and cloche^s5 embryo extracts were analyzed by western blotting with anti-αA-crystallin antibody. (B) Zebrafish embryo (2-4 dpf) mRNA levels were analyzed by semi-quantitative RT-PCR. Wild-type versus cloche^s5 embryo gene expression is shown for αA-crystallin and γ-crystallin. ef1a was used as a loading control. (C) αA-crystallin overexpression prevents γ-crystallin insolubility. Zebrafish embryos (2.5 dpf) were lysed detergent-free in 20 mM Tris-HCl, pH 7.5, and centrifuged. Soluble (top) and insoluble (bottom) fractions were separated, and were each analyzed by western blot with anti-γ-crystallin antibody. Two independent experiments using cloche^s5 were performed.

• Supplemental Figure 3 -

  Fig. S3. Analysis of lens in crash&burn mutants. crash&burn are mutants in bmyb that are developmentally delayed. There were no cataracts (n=80) in these mutants, 2-4 dpf. Thus, developmental delay is insufficient to induce cataract formation.