Fig. 1

Fig. 1 Characterisation of the sunrise (sri) Mutant
(A) Sections through wild type and homozygous sri larval eyes at 5 dpf, showing variable severity from mildly reduced lens size to almost complete absence of lens and retinal malformation (*). le, lens; re, neural retina and retinal pigment epithelium (black layer); arrowhead, optic nerve.
(B) Sequence traces from pax6b homeodomain revealing the T to C mutation resulting in the L244P mutation. The G to A third position change two bases earlier is a polymorphism between the WIK and Tü strains.
(C) Expression of known pax6 targets, glucagon and insulin, in pancreas of wild type (wt) 5 dpf fish. Expression is maintained in sri despite the loss-of-function homeodomain missense mutation in Pax6b, the sole pax6 gene expressed in the pancreas.
(D–F) Functional analysis of the Pax6b^sri (L244P) protein in comparison with wild type Pax6b and Pax6a, using luciferase reporter assays in HeLa cells and EMSA.
(D) Pax6a and Pax6b proteins drive luciferase expression at comparable levels under the control of the P3 homeodomain promoter; the L244P mutant of Pax6b protein fails to activate P3.
(E) Pax6a and Pax6b proteins drive comparable luciferase expression levels through the CD19 paired domain target promoter; the L244P mutant of Pax6b protein has significantly reduced activity.
(F) Gel-retardation assay to analyse the binding capacity to homeodomain target binding sites P2 and P3 demonstrates reduced affinity of the L244P mutant protein compared to wild type (wt) Pax6b.
D, dimeric protein-bound; M, monomeric protein-bound; and F, free labeled target oligonucleotide. Protein concentrations used 0.5, 1.5, and 4.5 μM (lanes from left to right) with 5 nM oligonucleotides.