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Abstract #19776 Published in IGR 9-4
Embryonic expression of the optineurin (glaucoma) gene in different stages of mouse development
Rezaie T; Stoilov I; Sarfarazi MMolecular Vision 2007; 13: 1446-1450
PURPOSE: To analyze optineurin (Optn) gene expression in various embryonic stages of mouse development by whole mount in situ hybridization. METHODS: FVB/NcrlBR mouse embryos (10.5 and 13.5 dpc) were collected by timed breeding experiments. A 712 bp Optn cDNA fragment was amplified by PCR and cloned into a transcription vector pCR®II-TOPO®. Digoxigenin labeled sense and antisense RNA probes were generated by in vitro transcription. The labeled RNA probe was localized using an anti-digoxigenin antibody conjugated with alkaline phosphatase. Colorimetric detection was performed with substrate solution containing, 4-nitro-blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP). RESULTS: This study revealed that the developing eye represents a major expression site for Optn. At both 10.5 and 13.5 dpc a strong specific expression was detected in the outer layer of the optic cup (future pigment layer of the retina). This is in contrast to the expression of another glaucoma gene, Cyp1b1, the expression of which at this state is only limited to the inner (neural) layer of the optic cup (future nervous layer of the retina). Inspection of sections from the cephalic region of whole mounts also revealed limited Optn staining in the lens as well as in the optic nerve. A second Optn expression domain was detected at the base of the developing forelimb. The biological significance of this observation is not clear and remains to be determined. CONCLUSIONS: Eye and forelimb were identified as two major sites for expression of the Optn gene. These findings suggest that Optn expression is triggered during early stages of eye development. Expression of the Optn gene in ocular tissues during mouse embryogenesis correlates with the presence and distribution of the optineurin protein, as previously reported in adult ocular tissues. These findings are also in agreement with the predicted function of Optn protein in the eye and the role of its ortholog in human glaucoma. Further investigations are required to determine the molecular mechanisms of Optn in the developing murine forelimb.
Dr. M. Sarfarazi, University of Connecticut Health Center, Molecular Ophthalmic Genetics Laboratory, 263 Farmington Ave., Farmington, CT 06030-1110, USA. mansoor@neuron.uchc.edu
Classification:
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
5.1 Rodent (Part of: 5 Experimental glaucoma; animal models)