Abstract
Purpose: :
With its well characterized retina and developmental stages, Xenopus laevis is an important model for studying eye development. Despite the wealth of data attained over the last few decades, many questions still exist surrounding the Xenopus retina. Of particular interest is the Xenopus green rod, which contains a blue–sensitive SWS–2 visual pigment. SWS–2 visual pigments have many interesting attributes in amphibians. In particular, the tiger salamander was recently found to have a blue–sensitive SWS–2 visual pigment in both green rods and blue cones. In an attempt to characterize promoter elements that drive rod or cone specific opsin expression, a transgenic Xenopus model was created that uses the tiger salamander SWS–2 promoter to drive expression of green fluorescent protein (GFP) in Xenopus green rods. In order to use the model, we must first characterize Xenopus green rod development. While the red rod and red cone opsin are detectable by immunohistochemistry at stage 35, immunohistochemical analysis of premetamorphic transgenic Xenopus tadpoles has failed to detect green rods. Thus, we hypothesize that the green rod develops during metamorphosis, separate from the other Xenopus photoreceptors.
Methods: :
Xenopus green rod development was analyzed by detecting green rod opsin expression with immunohistochemistry (IHC) and reverse transcriptase polymerase chain reaction (RT–PCR) on embryos from stage 30 through adult. Ten micrometer paraffin sections were stained with a polyclonal antibody specific for the Xenopus green rod opsin (XGN). RT–PCR was performed on dissected retinas of successive embryo stages to identify the presence of Xenopus green rod opsin mRNA.
Results: :
Adult retina sections stained with XGN and examined by fluorescent microscopy identified green rods, and RT–PCR on adult retina cDNA revealed Xenopus green rod mRNA. IHC and RT–PCR analysis of stage 45 embryos failed to reveal green rod opsin or green rod opsin mRNA, respectively.
Conclusions: :
Analysis of adult retinas demonstrated the specificity of XGN to Xenopus green rod, and RT–PCR on adult retinas showed primer specificity for green rod opsin cDNA. Because RT–PCR and IHC on stage 45 retinas have failed to show green rod opsin expression, it appears that the green rod develops independently of the other Xenopus photoreceptors.
Keywords: photoreceptors • retinal development • gene/expression