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H. M. El-Hodiri, Y. Pan, R. I. Martinez-De Luna, S. Nekkalapudi, L. E. Kelly; The Retinal Homeobox (Rx) Gene Product Is Necessary for Normal Expression of Photoreceptor-Specific Genes and Photoreceptor Function. Invest. Ophthalmol. Vis. Sci. 2009;50(13):488.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal homeobox (Rx) gene product is necessary for eye specification and development, however little is known about the molecular details of its function. Rx interacts with the PCE-1 (photoreceptor conserved element-1) sequence, found in many photoreceptor-specific promoters and can activate artificial reporter gene constructs containing multiple PCE-1 elements. Here we explore the involvement of Rx in regulating transcription of photoreceptor genes such as rhodopsin and red cone opsin.
In situ hybridization was performed using sectioned embryos and digoxigenin-labelled antisense riboprobes. Xenopus opsin promoter - luciferase (XOP-Luc) reporter plasmids and candidate transcriptional regulators were introduced into two-celled Xenopus laevis embryos or cos7 tissue culture cells. Luciferase activity was measured in lysates prepared from injected mid-gastrula embryos or transfected cells. Chromatin immunoprecipitation (ChIP) was performed using Xenopus embryos injected with low amounts of RNA encoding myc-tagged Rx or myc tag alone and antibodies raised against the myc epitope tag or control IgG. ChIP products were identified and quantified by real-time PCR using primers designed to amplify portions of the XOP or red cone opsin. Rx expression was knocked down using a transgenic shRNA approach. Tadpole vision was tested using a behavioral assay.
We verified that Rx is co-expressed with rhodopsin and red cone opsin in maturing photoreceptors and demonstrate that Rx can bind to the rhodopsin and red cone opsin promoters in vivo. We also found that Rx can cooperate with the Xenopus analogs of Crx and Nrl, otx5b and XLMaf (respectively), to activate a Xenopus opsin promoter-dependent reporter. Further, we specifically knocked down Rx expression levels using an shRNA targeted to Xenous Rx gene products but not a mismatch control shRNA. We found that reduction of the level of Rx gene expression in vivo results in decreased levels of rhodopsin and red cone opsin expression. Finally, knockdown of Rx results in abnormal photoreceptor morphology and impaired visual function.
Our data suggests that Rx, in combination with other transcription factors, regulates opsin gene expression and is necessary for normal photoreceptor morphology and function.
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