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Emili E Delp, Sudha Swamynathan, Winston Kao, Shivalingappa K Swamynathan; Spatiotemporally regulated disruption of Krüppel-like factor-4 (Klf4) results in loss of mature corneal epithelial cell identity . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5646. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previously, we demonstrated that conditional disruption of Klf4 in developing ocular surface resulted in corneal epithelial fragility, stromal edema and loss of conjunctival goblet cells. However, Klf4-conditional null (Klf4CN) mice derived using Le-Cre transgene were unsuitable for studying Klf4 functions in mature cornea. Moreover, it was impossible to parse out direct effects of the absence of Klf4 in Klf4CN cornea from indirect effects of their defective adnexae. Also, hemizygous Le-Cre mice occasionally display defective corneas in the absence of LoxP sites, raising additional concerns. Here, we have addressed those concerns by spatiotemporally regulated disruption of Klf4 selectively in corneal epithelium.
Cre expression was induced by doxycycline treatment in ternary transgenic (Klf4f/f/Krt12-rtTA/Tet-O-Cre) mice to ablate Klf4 in mouse corneal epithelium (Klf4Δ/ΔCE). Corneal epithelial barrier function was tested by fluorescein uptake. QPCR, immunoblots and immunofluorescence were employed to quantify selected Klf4-target gene expression.
Klf4 expression was efficiently disrupted within four days of Cre induction in corneas of Klf4Δ/ΔCE mice. The number of epithelial cell layers and Ki67-positive cells increased in Klf4Δ/ΔCE corneas, which displayed swollen and rounded basal epithelial cells unlike the columnar wild-type counterparts. Expression of epithelial basement membrane laminin-332, desmosomal components Dsp and Dsg, and corneal epithelium-specific Krt12 was downregulated, while that of Krt17 and MMP9 was elevated in Klf4Δ/ΔCE corneal epithelium. Increased number of CD45-positive cells provided evidence of inflammatory response in Klf4Δ/ΔCE corneas.
Spatiotemporally regulated disruption of Klf4 in normally formed mature corneal epithelium results in loss of corneal epithelial cell identity and squamous metaplasia. These results taken together with our previous reports confirm that Klf4 is essential for both maturation and maintenance of corneal epithelial homeostasis.
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