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John D Gottsch, Shivakumar Vasanth, Allen O Eghrari, Edwin Oh, S Amer Riazuddin, Nicholas Katsanis; Characterization of Fuchs Corneal Dystrophy phenotype in a TCF8 knock-in mouse model. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4826. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported a missense mutation (p.Q840P) in TCF8 responsible for Fuchs Corneal Dystrophy (FCD) in a large multigenerational pedigree and a sporadic late-onset case. To further investigate the physiological characteristics of FCD in a model that closely mimics human ocular tissue, we developed a knock-in mouse model harboring the variant homologous to the human mutation in Tcf8. The following study was undertaken to characterize the FCD phenotype in the murine model.
Tcf8Q818P/Q818P knock-in mice were developed by constructing a targeting vector using BAC recombineering with Neo cassette for positive selection of embryonic stem cells. Contact confocal microscopy was conducted in corneas of wild-type (Wt) mice and mice heterozygous (Tcf8wt/Q818P) and homozygous (Tcf8Q818P/Q818P) for the pathological variant. Pachymetry was assessed with peak-to-peak signal intensity from confocal Z-scan with 5 micron intervals. Endothelial cell density was calculated using the center method. The extent of guttae on confocal microscopy was assessed by an independent reviewer on a 1-to-5 scale with reference images. We utilized paraffin-embedded corneal sections for histopathological and immunohistochemical analyses.
Targeted ES clones and germline transmission were confirmed by Southern blotting while the mutation was confirmed by Sanger sequencing following RT-PCR of Tcf8 mRNA. We conducted confocal microscopy of 48 mice ranging from 0.3 to 2.1 years old. Among 4-month old mice, confocal imaging of 14 eyes from Wt mice and 14 eyes from mice harboring the mutation (9 homozygous, 5 heterozygous) revealed significantly thicker values compared to Wt mice in homozygous (95 vs. 118 microns, p<0.01) and marginally in heterozygous (95 vs. 108 microns, p=0.06) corneas (p<0.001, WT compared to all mice bearing mutation). Endothelial cell count was decreased in heterozygous compared to Wt (2289 vs. 2498 cells/mm2, p<0.01) and homozygous (2289 vs. 2544, p=0.03) mice. Compared to Wt, mean guttate appearance score was significantly higher among homozygous (P=0.016) and marginally in heterozygous (p=0.057) mice. Corneal sections of 18-month-old homozygous Tcf8 mice show reduced COL4A3 and COL8A2 expression compared to WT littermates.
We demonstrate a knock-in mouse model with guttate appearance, endothelial cell loss, and increased corneal thickness, pathological findings consistent with human FCD.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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