Targeted Deletion of AP-2α Leads to Disruption in Corneal Epithelial Cell Integrity and Defects in the Corneal Stroma

Severe defects in vision are frequently associated with abnormalities of the cornea, a highly specialized transparent tissue responsible for the refraction of light.1 The cornea comprises three tissue layers: the outer stratified squamous epithelium, the inner endothelium, and the intermediate stroma, the latter of which is composed of keratocytes (resident fibroblasts) separated by tightly packed, regularly arranged collagen lamellae. During development, the cranial neural crest cells give rise to the cells of the corneal stroma and endothelium,2 whereas a region of the head ectoderm that is further defined as the lens placode gives rise to the corneal epithelium.3,4 In mice, the corneal epithelium originates as a two layered structure that, after eyelid opening at 2 weeks of age, expands into a stratified epithelium consisting of 8 to 10 cell layers.5,6 The genetic programs responsible for early development of the cornea are governed by multiple transcription factors that act in a combinatorial fashion. Of significance is the homeobox and paired-box containing Pax6 gene, a member of the Pax gene transcription factor family, which is expressed in the early lens placode and its derivatives and is essential for normal eye development.7,8 Mutations in Pax6 in humans and mice have been shown to lead to congenital defects that result in lens cataracts, microphthalmia (small eye), aniridia (iris defect), and multiple defects in the cornea.3,9–12 In addition to its necessity during embryonic development, Pax6 has been shown to be have a central role in regulating postnatal differentiation and maintenance of the adult cornea.11,12 Specifically, the corneal epithelium of heterozygous Pax6 Small eye (Sey+/−) mice with half the levels of Pax6, exhibit a reduced number of stratified layers and compromised cellular adhesion.12 After injury to the normal corneal epithelium, Pax6 expression is induced at the wound edge where it has been shown to activate the gelatinase B (matrix metalloproteinase [MMP]-9) promoter.13 In Sey+/− mice, corneal injury results in an increase in both inflammation and the rate of reepithelialization, compared with the effect in wild-type mice.14 These observations indicate that appropriate levels of Pax6 expression are necessary to suppress excess inflammation and control the rate of reepithelialization after corneal injury. It is well known that Pax6 does not act alone in regulating gene expression during early eye development and, likewise, in the postnatal cornea, Pax6 has been shown to act in combination with additional regulators to activate or repress gene promoters. One of these regulators is AP-2α (activating protein-2α) transcription factor, which has a striking overlap in expression pattern with Pax6 in the developing and adult eye.15 Specifically, in the adult corneal epithelium, AP-2α expression is confined to the more basally located cells of the corneal epithelium, similar to Pax6.15,16 Earlier studies revealed a role for AP-2α in corneal epithelial repair in that its expression is upregulated at the leading edge of the migrating epithelium after wounding, and binds to and activates the MMP-9 promoter.14,17,18 More recently, AP-2α has also been shown to interact directly with Pax6 and facilitate its binding to the MMP-9 promoter.14 Further evidence for the requirement of AP-2α in corneal differentiation has been provided by the finding that overexpression of the AP-2α gene in a corneal epithelial cell line resulted in dramatic changes in cell phenotype, including a clumping growth behavior indicative of differentiation, as well as a change in cell adhesion expression.16 Requirements for AP-2α in ocular development have been revealed through studies of AP-2α knockout (KO) and chimeric mice.15,19 AP-2α null mice exhibited multiple and complex ocular phenotypes ranging from anophthalmia (absence of eyes) to mutant eyes that exhibited an adhesion of the lens to the overlying surface ectoderm. Multiple defects in the developing optic cup (future retina) were also observed.15 In the AP-2α null mice no corneas developed; this deficiency was attributed to secondary tissue defects. A proportion of AP-2α chimeric mice, composed of a mixture of AP-2α wild-type and null cells, exhibited a defect in separation of the lens from the overlying ectoderm and as a result had a persistent adhesion between the lens and corneal epithelium.15,19 AP-2α-null cells, identified using a specific β-galactosidase cell lineage tracer, were localized in these regions. However, in the remaining part of the corneal epithelium, the AP-2α-null cells were excluded, and the corneal epithelium appeared relatively normal. Because of this finding and the fact that the AP-2α-null mice do not form a cornea at all, these mutant models could not be used to investigate the specific in vivo requirement(s) of AP-2α in postnatal corneal epithelial differentiation. In the present study, we created a conditional KO mouse model of AP-2α (Le-AP-2α mutants), using the Cre-loxP approach, to examine the requirement for AP-2α in corneal epithelial differentiation. AP-2α expression in the Le-AP-2α mutant corneal epithelium was completely abolished, whereas other non–lens-placode–derived tissues maintained normal levels of AP-2α. Prominent defects in corneal epithelial stratification, cell adhesion, and basement membrane deposition were observed in the Le-AP-2α mutant corneas. In addition, the corneal stroma in these mutants exhibited an abnormal phenotype, including an activation of the resident fibroblasts. Together, these data reveal a specific requirement for AP-2α in the normal postnatal differentiation of the corneal epithelium.