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Topic Overview:
The development of the ocular surface and structures, including the lens, cornea, conjunctiva, and eyelids, involves the complex interplay of genetic and environmental factors. In the mouse cornea—following eye opening around two weeks after birth—the 1-2 cell layered epithelium begins to proliferate and differentiate, eventually forming a 5-8 cell layered stratified squamous mature epithelium by 6 to 8 weeks of age. Although defects at any stage of morphogenesis, maturation, or maintenance of the cornea can result in severe visual impairment, relatively little is known about the numerous transcription factors that regulate this process.

Kruppel-like transcription factors KLF4 and KLF5 are among the most highly expressed transcription factors in the mouse cornea as well as vital organs like the heart, skin, and intestine. Since Klf4-null and Klf5-null knockout mice uniformly die in utero or within hours after birth, studying the role of KLF4 and KLF5 in postnatal ocular development poses a formidable challenge. Using the Cre/loxP recombination system, Dr. Swamynathan has created mice with conditional deletion of the Klf4 gene in surface ectoderm-derived eye tissue.  The Cre/loxP system takes advantage of the cre gene, which encodes a site-specific DNA recombinase capable of removing a DNA fragment flanked by loxP recognition sequences through site-specific recombination.  Breeding mice that express Cre protein in corneal tissue with mice that have loxP sites flanking Klf4 sequences generates a mouse lacking ocular surface expression of KLF4. The resultant Klf4 conditional null (Klf4CN) mouse is also viable and fertile.

Dr. Swamynathan’s work on Klf4 conditional null mice has elucidated the many functions of KLF4 in the ocular surface. The Klf4CN cornea displays epithelial fragility with excessive sloughing of superficial cells, resulting in the presence of only 3-4 cell layers in spite of increased cell proliferation, stromal edema, and a reduction in the number of endothelial cells, which are extensively vacuolated. The Klf4CN conjunctiva lacks goblet cells, the anterior cortical lens is spongy and vacuolated, and the iris is hyperplastic.  These observations demonstrate the critical role of KLF4 in post-natal morphogenesis and maturation of the ocular surface.

Dr. Swamynathan’s Klf4CN mouse has potential application as a disease model for a range of pathologic ophthalmological conditions like dry eye and ocular cicatricial pemphigoid. His future research strategies include studies on Klf5CN mice and the generation of Klf4 and Klf5 double conditional null mice, the latter of which will identify any overlapping and/or antagonistic effects of these factors at the level of gene expression.