Retinal expression of zebrafish six3.1 and its regulation by Pax6

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Abstract

Homologues of the homeobox genes sine oculis (so) and eyeless (ey) are important regulators of eye development in both vertebrates and invertebrates. A Drosophila paralogue of so, optix, is an orthologue of the vertebrate Six3 gene family. Our analysis of zebrafish six3.1 demonstrated retinal expression in two separate cell layers and the ciliary marginal zone. This pattern is consistent with the observations of Six3 in other vertebrates and indicates functional conservation. We studied the 5 flanking region of six3.1 and showed that separate enhancing elements are required for expression at different stages of eye development. This analysis also revealed specific binding of zebrafish Pax6.1 protein to an element required for six3.1 expression in ganglion cells. Furthermore, an enhancement of six3.1 transcription by Pax6.1 was observed by co-injection experiments. These results provide evidence for a direct regulatory interaction between vertebrate Pax6 and Six3 genes in eye development.

Section snippets

Materials and methods

Production of RNA. Templates for RNA synthesis were prepared by PCR amplification using cDNA for six3.1 (AF030280) and pax6.1 (X61389). Template for six3.1 antisense probe was amplified using T7 and SX10 (GCGATCAACAAGCATGAATCCATC). The whole pax6.1 cDNA was amplified using T7 and PAX6-SP6 F (ATTTAGGTGACACTATAGAATATCACGAGCAACACGGTTA, underlined part shows an incorporated SP6 sequence). Amplified PCR products were gel-purified and RNAs were produced using T7 (six3.1) or SP6 (pax6.1) RiboMAX Large

Results

Using DNA in situ hybridization probes retinal expression of six3.1 was not detected during later stages of eye development [10]. Since this observation was not consistent with the results reported for other vertebrate species we made further investigations using RNA whole-mount in situ hybridization. By this approach we detected six3.1 transcripts in the differentiating retina from 32 hpf. At this stage a thin layer of six3.1 staining is located in a part of the retinal neuroepithelium (RNE)

Discussion

The early expression pattern of zebrafish six3.1 shows clear similarities to vertebrate orthologues (mouse, human, chicken, Xenopus, and medaka) and the zebrafish paralogues six3.2 and six7[6], [22]. A particularly close relationship has been demonstrated for the two zebrafish genes six3.1 and six3.2 possibly reflecting an extra genome duplication in fish [5]. Although the early expression patterns are similar for these two zebrafish genes, the later retinal expression is quite different.

Acknowledgements

We thank Dr. Thomas Becker for providing zebrafish albino embryos and β-gal antibody and Dr. Øyvind Drivenes and Dr. Ståle Ellingsen for helpful discussions and scientific/technical advice. This work was funded by the Norwegian Research Council and Norwegian Cancer Society.

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    Present address: Institute of Marine Research, P.O. Box 1870, Bergen N-5024, Norway.

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