Spatiotemporally Regulated Ablation of Klf4 in Adult Mouse Corneal Epithelial Cells Results in Altered Epithelial Cell Identity and Disrupted Homeostasis

The cornea, comprised of an outer stratified squamous epithelium, central collagenous stroma, and inner endothelial monolayer, is essential for clear vision. The corneal epithelium, being the most anterior tissue of the eye, provides the first line of defense against noxious chemical, biological, and physical stimuli.1 Loss of corneal epithelial integrity is associated with debilitating ocular surface disorders, such as dry eye, ocular cicatricial pemphigoid, and Meesmann's dystrophy.1–3 The newborn mouse corneal epithelium consists of one to two cell layers of unstratified cells, which divide and differentiate in a highly coordinated process to form a three- to four-cell layered stratified epithelium by postnatal day 12 (PN12) concurrent with eyelid opening.4,5 The adult mouse corneal epithelium consists of six to eight stratified cell layers that express cornea-specific keratin-12 and adherens junctions in all layers, tight junction proteins at the superficial layers, desmosomes in the spinous cell layers, gap junctions, and hemidesmosomes in the basal cell layers.4–14 Mature corneal epithelial cells undergo recurring cycles of self-renewal, during which progenitor/stem cells residing in the limbus provide a source of transient amplifying cells that proliferate as they migrate centripetally, subsequently differentiating into mature epithelial cells as they move up from the basal layer to form wing cell and superficial cell layers to replace those lost through tissue turnover.15–17 Though several transcription factors, including Klf4, Klf5, Pax6, AP2α, HMGN1, Cited2, basonuclin, Sp1, AP1, NF1, EHF, and Oct1, are known to regulate corneal development, our understanding of the gene regulatory networks that control proper maintenance of the adult corneal epithelium remains incomplete.18–36 Kruppel-like factor-4 (Klf4), which regulates a diverse array of cellular events, including proliferation, differentiation, stem cell maintenance, development, and apoptosis, is one of the most highly expressed transcription factors in the mouse cornea.37–43 Our previous studies demonstrated that Klf4 has an integral role in the mouse ocular surface maturation.20,41,44–47 Though Klf4 conditional null (Klf4CN) mice, derived by mating Klf4LoxP/LoxP (Klf4f/f) with Klf4f/f/Le-Cre/- mice, wherein Klf4 was disrupted from embryonic day 10 (E10), were helpful for revealing the role of Klf4 in corneal maturation, they were not suitable for addressing the role of Klf4 in maintenance of the adult mouse cornea,48,49 As Le-Cre also is expressed in the eyelid epidermal epithelium, lacrimal glands, meibomian glands, and the conjunctival epithelium, it was impossible to parse out the direct effects of the absence of Klf4 in corneal epithelium from indirect effects of the defective surrounding tissues in Klf4CN mice.20,45,50,51 Moreover, hemizygous Le-Cre transgenic mice that we used to generate Klf4CN mice occasionally develop eye abnormalities on some genetic backgrounds even in the absence of LoxP sites, raising additional concerns about the validity of our findings related to corneal functions of Klf4.52 In this study, we have addressed those concerns by exploiting doxycycline-inducible expression of Cre recombinase in a ternary transgenic mouse line53,54 to selectively delete Klf4 within the corneal epithelium in a precisely spatiotemporally regulated manner. Based on the data obtained from this approach, we reported that Klf4 is essential for maintenance of the adult corneal epithelial cell identity and homeostasis.