(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.

(A–D) Phenotype elicited using pax6b morpholinos: (A) Uninjected wild type fish at 48 hpf; fish injected with the same amount of a control scrambled morpholino also produced no effect.
(B) Substantially reduced eye size and generally delayed development are observed by 48 hpf in response to injection of either of the two pax6b morpholinos.
(C and D) At 32 hpf reduction in eye size is observed (white dotted outlines) in otherwise normal looking embryos.
(E) The effects of injecting capped mRNA at levels that generally rescue the phenotype illustrating the spectrum of phenotypes to bilateral anophthalmia, observed when pax6b capped mRNA is injected at levels generally used for phenotype rescue experiments in zebrafish.

(A) Vista plot of the region around the highly conserved region E60+, located 47 kb downstream from the PAX6 P1 promoter. Human sequence is used as the base for pairwise comparison with pax6 loci from mouse, Xenopus tropicalis, and zebrafish (pax6a and pax6b). Fragment E60B_UCS contains an ultraconserved element that will be described elsewhere and is used here as an anchor point to allow localisation of the conserved/non-conserved E60A element from zebrafish pax6a and pax6b. A fragment containing the human E60A element was cloned into an hsp68-LacZ reporter construct to generate transient transgenic embryos. The equivalent regions from the zebrafish pax6a and pax6b loci were cloned into a Tol2 transposon based vector containing a cFos minimal promoter GFP reporter cassette [53] and used to produce transgenic zebrafish.
(B–E) Reporter expression of the human E60A construct was observed in optic cup, telencephalon, and prosomere P3 region of the diencephalon in embryos of E9.5 (B); E10.5 (C and D); and E11.5 (E). Expression (ectopic) in the limbs is ascribed to the genomic integration site of the construct.
(F) A similar GFP reporter expression pattern is seen in transgenic zebrafish embryos with the E60A region from the pax6a locus, with GFP expression observed in the optic cup and forebrain.
(G) No reporter expression is seen with the construct containing the E60A region from locus pax6b.
(H) Frontal view of a transgenic zebrafish with the pax6a E60A element, showing expression in telencephalon and optic cup.
(I) Frontal view of a pax6b E60A transgenic fish showing a lack of specific expression. T, telencephalon; OC, optic cup; P3, prosomere P3 of the diencephalon.

pax6a expression is shown in (A) and (C) at 24 hpf; (A) lateral view showing expression in eye, telencephalon, diencephalon, hind brain, and neural tube (C) dorsal view; (E) and (G) lateral and dorsal view at 32 hpf, showing continuing pattern of expression, (I) Vibratome coronal section of wholemount stained embryo expressing pax6a in telencephalon (arrowed), diencephalon, retina and lens. (B) pax6b expression is seen in lateral view showing eye and optic tectum at 24 hpf, pancreas expression is highlighted using black triangles; (D) dorsal view at 24 hpf. (F) and (H) lateral and dorsal. Increasing hindbrain and neural tube expression of pax6b is revealed by 32 hpf embryos. (J) predominantly eye expression of pa6b is seen in coronal section at 32 hpf.

(A) Synteny relationships of pax6 with its nearest neighbours. In mammals pax6 is flanked by rcn1 and elp4 genes with wt1 located further upstream. Horizontal arrows show direction of transcription. Vertical arrows indicate the positions of a subset of the pax6 cis-regulatory elements, many located downstream of pax6, within introns of the adjacent elp4 gene. Dotted arrows denote partially conserved elements. Zebrafish LG25 retains pax6a synteny with wt1a and with the majority of pax6 cis-regulatory elements, but rcn1 and elp4 coding exons have been lost. On LG7 the pax6b locus and rcn1 and elp4 exons have been retained, but many of the pax6b control elements, as well as the wt1 homolog, have been lost.
(B) PIP plot comparing the PAX6 locus in human with mouse, Xenopus and zebrafish pax6a and pax6b. Exons are shown in red, untranslated regions in pink, conserved cis-regulatory elements are shown in green, while absence of significant sequence conservation is indicated by a light blue box, and promoter P0 is demarcated in purple. The cis-regulatory elements discussed in the text are surrounded by open red rectangles. Enhancers: P, pancreas; T, telencephalon; NRE, neuroretina; 7CE2, diencephalon; E60B, ultraconserved enhancer with multiple brain specific functions (D. McBride, D. A. Kleinjan, and V. van Heyningen unpublished data); E60A, optic cup, early telencephalon and diencephalon P3 region.

(A) PIP plot showing the conservation levels in the Pax6 upstream regions of mouse, Xenopus tropicalis, and the two zebrafish co-orthologues using the human gene as base sequence. The position of the pancreas/ectodermal (P/EE) and the P0 promoter regions used in the construction of the reporter transgenes are shown.
(B) Schematic representation of the Tol2-2way system to create combinatorial constructs for Tol2 mediated zebrafish transgenesis.
(C) Expression analysis of pax6a and pax6b shown by RNA in situ analysis with alkaline phosphatase staining at 28 hpf (top). YFP expression in P/EE(A)-P0(A)-YFPpA (left) and P/EE(B)-P0(B)-YFPpA (right) transgenic fish at 28 hpf shown, using in situ analysis for YFP mRNA. 56 hpf fluorescence images of YFP expression in transgenic lines for the same two constructs are shown on the bottom row.
(D) Frequency of expression in different sites in transient transgenic zebrafish with the four combinations of P/EE enhancer and P0 promoter from the two pax6 loci. T, telencephalon; MHB, midbrain hindbrain boundary; HB, hindbrain; P, pancreas; NT, neural tube.