Fig. 5

Transgenic analysis of the elephant shark Pα promoter/neuroretinal enhancer (NRE) element.

A) PIP plot of the Pax6 intragenic region shows the conservation at the P±/NRE in intron 4 of mammalian Pax6 in both Pax6 loci in elephant shark. The NRE(Cm6.2) element is the only conserved non-exonic sequence between the Pax6.1 and Pax6.2 loci. To allow comparison with the mouse neuroretinal enhancer (NRE) LacZ reporter transgenic mice were made with the Cm6.2 element. (B-G) LacZ staining is seen in the neuroretina of these mice from E10.5 in a pattern that is highly similar to expression driven by the mouse element. B) No expression is seen at E9.5. C) Expression starts at E10.5 at the lateral sides of the developing eyes (E). Expression in the midbrain (M) is due to site of integration and is not seen in other lines. D) By E12.5 expression has widened to include the whole retina. The inset shows a larger view of the eye. E) Cross section through a E12.5 embryo head shows staining in the neuroretina (NR) and optic nerve. F) Expression continues at E14.5 in the neuroretina, and G) is maintained at E17.5 in the retina but not in the lens. (H-J) To test for functional conservation of the elephant shark Pax6.1 and Pax6.2 NRE we assessed the elements in zebrafish transgenics using the dual colour reporter system. H) The NRE element from the elephant shark Pax6.1 locus drives GFP reporter expression in the retina of transgenic zebrafish at 48 hpf. I) The NRE element from the elephant shark Pax6.2 locus similarly drives mCherry in the neuroretina at 48 hpf. J) The merged image shows functional equivalence of the Cm6.1 and Cm6.2 NRE elements in driving reporter expression in the zebrafish retina. NR, neuroretina, L, lens.