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Minh-Thanh T. Nguyen, Gracia Y. Ng, Leah A. Rosenfeldt, Shannon R. Balser, Ruth Ashery-Padan, Chia-yang Liu, Winston W. Kao; Ablation of Pax6 in the Basal Corneal Epithelial Cells Changes the Rate of Desquamation. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5247.
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To determine Pax6 cell autonomous function in the proliferation rate and desquamation of corneal epithelial cells.
Krt14-rtTA (K14R), tetO-Cre (TC), and ROSAmTmG (mTG) transgenic mice were crossed to obtain triple transgenic K14R/TC/mTG mice; in a second strain, Pax6flox/flox transgenic mice were also crossed, resulting in quadruple-transgenic mice (K14R/TC/mTG/Pax6f/f). Upon doxycycline induction, both mTG and Pax6flox alleles are converted to yield of mTΔG (mG, change from red to green fluorescence) and Pax6Δ alleles in cells expressing Cre recombinase. Pulse induction was performed by a single dose of doxy at 80 μg/g body mass I.P. injection two hours after the mice were injected with BrdU (120 μg/g body mass). Corneas from both strains of mice were collected 1, 2, 3, 5, 7, 10, 14, and 21 days after induction; these samples were stained with anti-BrdU antibody and DAPI. Z-stack fluorescence images were captured using Zeiss Apotome in central, peripheral and limbal regions. Changes in adheren junctional proteins such as Δ-catenin, E-cadherin, β-catenin and some integrins such as α4 and β1 were also examined.
Preliminary data showed Pax6cbeΔ/ cbeΔ/mGcbe cells moved higher up through the corneal layers in a shorter amount of time compared to normal corneal epithelial cells expressing Pax6. By day 7, most green cells in the Pax6cbeΔ/ cbeΔ/mGcbe cells have already reached the upper wing and/or superficial cells whereas majority green cells in mGcbe have just moved out of suprabasal cells. There appeared to be a difference in proliferation rates between corneas with Pax6 ablated and control corneas. Cellular adheren junctional proteins seemed to be altered in the Pax6cbeΔ/cbeΔ/mGcbe corneas as compared to the mGcbe corneal epithelium.
Pax6 expression in corneal basal epithelial cells is needed to regulate the rate of normal physiological upward cell movement and desquamation through its effects on adheren junctional complex. The mGcbe and Pax6cbeΔ/mGcbe animal models may be useful in studying the function of Pax6 in corneal epithelial cell stratification.
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