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X. C. Zhao, S. Yoshikawa, L. D. Carter-Dawson; Zebrafish Ocular Tissue-Specific Promoters. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1692.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported cloning and characterization of promoters and enhancers for the cornea and the connective tissue of the anterior angle. To study the function of ocular tissues and their interactions for normal vision, we cloned and analyzed additional promoters that specifically express in the lens, retinal ganglion cells (RGC), and photoreceptors.
The putative promoter regions were cloned from three zebrafish genes, atonal homolog 7 (atoh7), cone-rod homeobox (crx), and retinal homeobox gene 1 (rx1), into a Tol2-based green fluorescence protein (GFP) expression vector. The mouse crystallin beta B1 (cbb1) promoter was cloned into the same expression vector. After verifying DNA sequences, these expression vectors were used to establish stable transgenic lines. GFP-positive embryos were observed under a fluorescence dissecting microscope, photographed, and sectioned for confocol imaging to determine the tissue specificity of these promoters.
Photoreceptor-specific GFP expression was first detected on the positive embryos of the rx1 lines at 4 days postfertilization (dpf) and confirmed by confocol analysis on embryos at 6 dpf. These results indicated that the 2.9 kilobase (Kb) DNA from the rx1 upstream region contained the functional promoter and enhancers for photoreceptor-specific expression. In the atoh7 line, GFP was first detected in the retina at 2 dpf. Confocol analysis showed that almost all RGCs expressed GFP at 4 dpf, suggesting the atoh7 promoter (1 Kb) contained all required elements for RGC-specific expression. GFP signials were also seen in the Inner plexiform layer. Transgenic embryos of the crx promoter (1.5 Kb) expressed GFP in the retina at 4 dpf. On sections of 6 dpf embryos, strong GFP signals were in the inner plexiform layer and about 50% of RGCs, but relatively weak GFP in the photoreceptors. A few amacrine cells very close to the Inner plexiform layer were GFP positive. The mouse cbb1 promoter (0.5 Kb) expressed very strong GFP in the lens starting at 3 dpf.
Three ocular tissue-specific promoters (the lens, photoreceptors, and retinal ganglion cells) were identified. These promoters should be very useful genetic tools for analyzing intercellular and tissue interactions and for studying eye diseases in zebrafish.
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