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A. M. Verdoni, A. Ikeda, W. W. Kao, S. Ikeda; Conditional Ablation of Srf in the Corneal Epithelium Rescues the Abnormalities in corn1 Mice. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5199.
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Corneal disease-1 (Dstncorn1) mice are deficient for destrin, which depolymerizes filamentous actin (F-actin) into its monomeric form. Dstncorn1 mice exhibit accumulation of F-actin in the corneal epithelial cells, recruitment of inflammatory cells to the cornea, epithelial cell hyperproliferation, and stromal neovascularization. In a previous study, we identified that serum response factor (Srf) and 53 of its target genes are upregulated in the cornea of Dstncorn1 mice. The purpose of this study was to determine the role of SRF in the development of Dstncorn1 phenotypes through conditional gene ablation specific to the corneal epithelial cells.
Mice carrying the Dstncorn1 allele, an Srf allele flanked by loxP sites (Srff), a corneal epithelial specific reverse tetracycline transcriptional activator (Krt12rtTA), and cre recombinase downstream of a tetracycline responsive element (TetO-cre) were generated. Doxycycline was injected intraperitoneally in mice every other day to induce cre recombinase expression for specified time periods.Whole mount immunohistochemistry was used to assess the levels of F-actin and neovascularization. Immunohistochemistry was performed on sections to identify proliferating cells in the epithelium and inflammatory cells in the stroma.
Injection of Doxycycline for 30 days demonstrated that conditional ablation of SRF expression in the corneal epithelium rescues the abnormalities observed in Dstncorn1 mice. In mice with the genotype Dstncorn1/corn1 Srff/f Krt12rtTA/+ TetO-cre+, the level of F-actin is greatly reduced, as is the level of inflammatory cell infiltration. The number of proliferating cells is also markedly decreased, and the pattern of cell proliferation resembles that of a normal cornea. Neovascularization is also less evident.
This study demonstrated that abnormal activation of SRF is primarily responsible for the corneal abnormalities observed in Dstncorn1 mice. In particular, Srf was found to mediate the F-actin accumulation, inflammatory cell recruitment, hyperproliferation, and neovascularization phenotypes in Dstncorn1 mice. This study also showed that these abnormalities are due to SRF expression in the corneal epithelial cells, and demonstrated that the stromal phenotypes, inflammation and neovascularization, occur due to altered gene expression in the corneal epithelium of Dstncorn1 mice.
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