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R. I. Martinez, H. M. El-Hodiri; Silencing the Retinal Homeobox (Rx) gene impairs retinal regeneration in pre-metamorphic Xenopus laevis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1274.
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The retinal homeobox gene (Rx) is essential for eye development and retinal progenitor cell (RPC) maintenance. Loss of Rx function leads to a lack of eye development in a variety of species. Prior studies in our laboratory suggested that, in addition to being necessary during eye development, Rx might also be necessary during retinal regeneration. We found that after injury to the retina in pre-metamorphic X. laevis, the wound is repopulated with retinal progenitor cells (RPCs) that express Rx. The purpose of this study is to determine the function of Rx in the regenerating retina.
Tadpoles transgenic for a shRNA designed to target the two Rx genes in X. laevis were generated by the intracytosolic sperm injection (ICSI) method. Tadpoles transgenic for a control shRNA that does not target Rx were also generated as controls. Regeneration was induced in these tadpoles by resection of the nasal-dorsal segment of the retina at stage 44. Regeneration was analyzed by histology. In situ hybridization was performed on sectioned retinas to examine the expression of Rx and Pax6. Quantitative PCR was performed to examine Rx expression levels in the transgenic Rx shRNA tadpoles.
Both Rx genes are partially silenced in Rx shRNA tadpoles but not in control shRNA tadpoles. After partial retinal resection, we observed that regeneration was impaired in Rx shRNA tadpoles. We previously determined that the wound has closed and is repopulated with RPCs at 9 days post-resection. In some Rx shRNA transgenics, the RPE has not yet closed around the wound and appears disorganized at 9 days post-resection. In addition, the RPCs that repopulate the wound are present, but have a rounded morphology, instead of the typical elongated RPCs observed in control tadpoles. We found that Rx and Pax 6 expression levels are markedly reduced in the RPCs that repopulate the wound. We have previously observed RPE-65 immunoreactivity in RPCs repopulating the wound in 38% of regenerating embryos. We did not observe RPE-65 immunoreactivity in the wounds of Rx shRNA transgenic tadpoles, suggesting that RPE transdifferentiation may be inhibited.
Impaired retinal regeneration was observed in Rx shRNA transgenic tadpoles, suggesting that Rx plays an important function during regeneration. A possible mechanism by which decreased Rx function affects retinal regeneration may be by interfering with RPE transdifferentiation.
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