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J. M. Wilson, M. W. King, A. L. Mescher, A. W. Neff, T. L. Belecky-Adams; Ddx39 in Developing and Regenerating Xenopus Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5703.
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Proteins that regulate the balance between proliferation and differentiation are logical candidates for manipulating regeneration in the nervous system. Ddx39 and Bat1, members of the RNA helicase family of DEAD-box proteins, play antagonistic roles towards each other in regulating this balance. The goal of this study is to determine the expression patterns of Ddx39 in the proliferation of developing and regenerating retina and determine if changes in the levels of Ddx39 will modify the number of Xenopus laevis retinal stem cells.
In situ hybridization was used to determine the expression patterns of Ddx39 in Nieuwkoop and Faber (NF) stages 15-58 and in FGF2-mediated regenerated retinae 2, 4, 7, 14, and 30 days post-retinectomy at NF stage 53. GFP and Ddx39 were cloned into the inducible pTet-On system to allow us to induce expression at different stages of development and/or regeneration. Expression vectors were lipofected into NF stage 24 optic vesicles using DOTAP (Roche, Indianapolis, IN) and induced with a range of doxycycline from 5-50ug/ml added directly to rearing water. Treatment of retinal tissue with doxycycline, as well as the effects of controlling Ddx39 over-expression was analyzed using immunohistochemistry and in situ hybridization from stage 24 to 41.
Ddx39 expression was localized to the region of the developing retina known as the ciliary marginal zone (CMZ) between NF stages 37-58. A comparison with other CMZ markers showed that Ddx39 was found in proliferating cells within the CMZ. In preparation for overexpression studies, the effect of doxycycline in 0.1X MMR or 0.4X Holtfreter’s on normal retinal development was studied. The doxycycline was added at 0, 5, 10, 25, and 50ug/ml and the tadpoles reared at 25°C until they reached NF stage 41. No differences were noted in the weights of embryos or expression patterns of cell type specific markers in cryosections of retinas from embryos reared with or without doxycycline.
1) Expression patterns of Ddx39 in development and regeneration were consistent with a possible role for this protein in the maintenance of stem cells in the retina. 2) Doxycycline does not appear to affect development of the retina at the concentrations used. 3) An inducible system coupled with ability of Xenopus laevis to regenerate retina make it an ideal system for studying retinal regeneration.
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