Xenopus Rx-L Gene Is Involved in Photoreceptor Development

Purpose: 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. Methods: 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. Results: 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. Conclusions: Our results suggest that the function of Rx-L is to regulate rod and cone development by activating photoreceptor-specific gene expression.