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. 2019 Dec 20;9:19559. doi: 10.1038/s41598-019-56085-8

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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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.