Photoreceptor Degeneration Caused by a Rhodopsin–GFP Transgene in Xenopus Laevis

Abstract

Abstract: : Purpose:We previously showed that P23H rhodopsin transgenes induce photoreceptor degeneration in Xenopus laevis as they do in mammals. The purpose of this study was: 1) to examine the cell–autonomy of transgene induced photoreceptor degeneration in Xenopus laevis, and 2) to determine the feasibility of monitoring the loss of photoreceptors externally in live animals. Methods: A rhodopsin–GFP (Rho–GFP) fusion protein transgene was made by inserting GFP at the C–terminus of the Xenopus laevis rhodopsin coding sequence within a 8 kb genomic rhodopsin clone using recombineering. Transgenic animals were made by REMI–nuclear transfer. Select animals were raised to sexual maturity and used for breeding. F1 progenies from transgenic and wild type animals were used for eye grafting experiments and in external measurements of photoreceptor degeneration. Reciprocal grafts of dorsal retina halves were produced between transgene positive F1s and wild–type embryos and the resulting tadpoles were sacrificed on day 10. Eye sections were stained with the 1D4 rhodopsin antibody. External measurements of photoreceptor loss were obtained by daily imaging of fluorescence emitted through the lens using subsaturating exposure times and a custom script written for IPlab software. Results: In some animals expressing the Rho–GFP trangenes severe and progressive retina degeneration was observed. This degeneration was most evident in the older central part of the retina and was accompanied by mislocalization of the Rho–GFP protein. Reciprocal grafting of retinas between transgenic and wildtype animals demonstrated that the mechanism of photoreceptor degeneration in this model is mainly cell–autonomous. External measurements of transgene fluorescence emitted through the lens were shown to be a reliable measure of the internal loss of photoreceptors. Conclusions: Expressing a Rho–GFP fusion protein by means of a genomic transgene in Xenopus laevis rod photoreceptors induces photoreceptor degeneration that is accompanied by mislocalization of rhodopsin. The rod photoreceptor degeneration induced by overexpressing Rho–GFP is mainly cell–autonomous. The progression of retina degeneration in Xenopus laevis can be monitored externally and will be used to screen therapeutic agents that slow or prevent retina degeneration.

Keywords: retinal degenerations: cell biology • cell-cell communication • cell death/apoptosis