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T. Rezaie, I. Stoilov, M. Sarfarazi; Confined Ocular Expression of Optineurin (Optn)During Mouse Eye Development by Whole–Mount In Situ Hybridization . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4446.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To study expression pattern of Optineurin (Optn), an adult–onset primary open angle glaucoma gene, during different developmental stages of mouse embryos. Methods: Non–isotopic whole–mount In Situ hybridization was used for detection of Optn–mRNA expression sites during mouse development. Embryos from FVB/NCrlBR strain were used. A PCR amplified Optn cDNA fragment was cloned into a transcription vector. Digoxigenin labeled sense and antisense RNA probes were generated by in vitro transcription of linearized plasmids. The labeled RNA was localized using an anti–digoxigenin antibody conjugated with alkaline phosphatase. Colorimetric detection was performed with NBT/BCIP substrate solution. Results: Previously we mapped, cloned and identified the genomic structure of mouse Optineurin gene. Our previous studies demonstrated expression of Optn transcripts in adult mouse tissues by Northern blotting. Our immunohistochemistry studies determined ocular localization of the optineurin protein in adult mouse eye tissues and optic nerve. By RT–PCR we were able to detect Optn mRNA in 7– and 11–days old mouse embryos. In the present study we used whole mount In Situ hybridization in order to facilitate a more precise and three–dimensional localization of Optn transcript during various stages of mouse development. We studied site–specific pattern of Optn expression during developmental stages of 9.0, 9.5, 10.5, 12.5, and 13.0 dpc (days post conception). Strong Optn expression domains were identified only in the developing eye as well as at the base of the forelimb bud. Inspection of sections from cephalic region of the whole mounts revealed Optn expression in the outer layers of the optic cup (future pigment layer of retina). Additional tissue sections from these embryos are currently under study. Conclusions: Our study indicates that: 1)–Optn expression is triggered early during the eye development; 2)–Optn expression is localized in tissues corresponding to future pigment layers of the retina and; 3)–eye is a major site of Optn expression in the developing embryo. Supported by NIH (EY–09947) and AHAF National Glaucoma Research.
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