The lamprey genome contains three Pax6 genes. (a) The three Pax6 loci in the Japanese lamprey, L. japonicum. Lamprey Pax6 genes are named Pax6α, Pax6β and Pax6γ. The BAC clones sequenced are shown below. LjPax6γ resides on a short (66 kb) scaffold containing no other genes. (b) Gene synteny comparison of the three Japanese lamprey (Jlamprey) and sea lamprey Pax6 loci with Pax6 loci from selected gnathostomes.

Protein sequences and expression patterns of the Japanese lamprey Pax6 genes. (a) Comparison of the amino acid sequences of LjPax6α, LjPax6β and LjPax6γ with human (Hs) PAX6. All three LjPax6 genes encode a highly conserved paired domain (solid black line), in contrast to known Pax6.2 (also referred to as Pax10¹⁴) genes which lack the sequences coding for this domain, as well as a highly conserved homeodomain (dotted grey line) and C-terminal transactivation domain. No evidence was found for the presence of the alternative exon 5a in the lamprey genes. The positions of the exon boundaries (black arrow head, phase 0 intron; open arrow head, phase 1 intron) are conserved between the human and lamprey genes. (b) qRT-PCR analysis using a panel of adult lamprey tissues showing the tissue-specific expression pattern of the LjPax6 genes. All three genes are highly expressed in the eye and brain, with lower and variable expression in other tissues.

Maximum Likelihood tree of vertebrate Pax6 genes. Phylogenetic analysis of the lamprey Pax6 genes with Pax6, Pax4, Pax3 and Pax7 genes from several other chordate species. The Pax3/7 clade was specified as the outgroup. The phylogram is shown on the left and its cladogram on the right. The tree highlights clustering of the three lamprey genes with Pax6 genes from other chordates thereby indicating that they are indeed lamprey Pax6 genes.

An ancient vertebrate conserved non-coding element is present in the lamprey Pax6β locus. (a) VISTA plot of the SLAGAN alignment of the LjPax6β locus, against the two Pax6 loci from elephant shark and human, as well as the amphioxus locus. Note that there is no PAX6 gene in the virtual ‘human_6.2’ locus. Sequence similarity outside of exons was seen for only a single element, homologous to the PAX6 neuroretina enhancer (NRE). (b) Conservation at the core sequence of the NRE element. (c) Transgenic zebrafish assay of the LjPax6β putative NRE element. The Lj_NRE element was cloned in front of a minimal promoter-eGFP cassette and used to generate stable transgenic fish. Embryos at 24, 36, 48 and 72 hours post fertilisation (hpf) show highly specific and consistent GFP signal in the neuroretina of the developing eye. Ectopic expression in the heart due to site of integration effect was seen in one of the transgenic lines. Embryos were imaged from the lateral side (24 hpf and inset 72 hpf showing a close-up of the eye) or ventral side (36 hpf, 48 hpf and 72 hpf) with the fluorescent confocal signal overlaid on a brightfield view. L, lens; NR, neuroretina; h, heart.