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Y. Pan, S. Nekkalapudi, L.E. Kelly, H.M. El–Hodiri; Xenopus Rx–L Gene Is Involved in Photoreceptor Development . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5409.
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© ARVO (1962-2015); The Authors (2016-present)
The retinal homeobox gene (Rx) is one of the earliest expressed eye genes and plays a very important role in eye development. Loss of Rx function results in severe defects in eye development in several species. We have identified a Rx–like (Rx–L) gene from Xenopus laevis. Rx–L shares near–identity with Rx at the homeodomain and OAR domain but lacks an octapeptide motif. Rx–like genes have also been isolated from chicken (RaxL) and humans (QRX). The function of Rx–L gene in retinal development is unknown, and no loss of function study has been performed. The purpose of this study is to use a loss–of–function approach to investigate the function of Rx–L in retinal development.
Xenopus opsin promoter – luciferase (XOP–Luc) DNA and RNAs encoding transcriptional regulators were microinjected into two–cell Xenopus laevis embryos. Luciferase activity was measured in lysates prepared from injected mid–gastrula embryos. Mobility shift assays (EMSA) were performed using in vitro translated proteins, radiolabeled PCE–1 probe, and oligonucleotide competitors. RT–PCR was performed using cDNA from whole embryos at various stages. Expression patterns were analyzed by in situ hybridization using whole or sectioned embryos and digoxigenin–labelled antisense riboprobes. Morpholino targeting specific gene was co–injected with GFP into four cell stage embryos and stage 41 or 45 embryos were paraffin embedded and sectioned at 8 µm and probed by in situ hybridization or immunohistochemistry.
Rx–L expression is first detected at neural tube stages, later than the onset of Rx expression at neural plate stages, and continues through retinal maturation. Both Rx–L and Rx can bind a PCE–1 (photoreceptor conserved element) DNA element, found in the promoter regions of all known photoreceptor genes. Rx–L functions as a stronger transcriptional activator than Rx in reporter gene assays. Antisense morpholino–mediated knockdown of Rx–L expression results in a decrease in the transcription level of both rhodopsin and red cone opsin in Xenopus and also a decrease in the number of both rods and cones and a shortening of rod and cone outer segments.
Our results suggest that the function of Rx–L is to regulate rod and cone development by activating photoreceptor–specific gene expression.
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