Disruption of mouse corneal epithelial differentiation by conditional inactivation of pnn.

A smooth, intact, and healthy corneal epithelium is requisite for normal vision. The stratified squamous non-keratinizing epithelium of the cornea accounts for approximately 80% of the refractive power of the anterior eye. In addition, this epithelium is at the interface with the external environment; thus, it must resist pressure and provide a barrier to fluid loss and pathogen entrance, all while maintaining maximal transparency.1 The specific identity and differentiated qualities of the corneal epithelium afford many properties that are requisite for its role at the anterior surface of the eye. Indeed, there exist numerous examples of ocular surface diseases whereby the corneal epithelial quality or differentiative qualities are lost, and significant anterior eye physiological perturbations and dramatic vision loss result. Many of these pathologic states result in the transformation of the bone fide corneal epithelium to a keratinized epithelium, which is a vastly inadequate epithelium for the maintenance of the stable tear film, corneal surface barrier, and avascular corneal stroma. Thus, elucidation of the molecular details pertaining to establishment and maintenance of the corneal epithelia is central to the design of new therapies to impact the maintenance and repair of the corneal epithelial phenotype and the corneal epithelial barrier. It is now becoming apparent that the maintenance of the specific cell phenotype and physiology of the anterior eye remains dependent on sustained regulation of some of the same molecular pathways and mechanisms that play such important roles in development.2–4 However, a relatively small number of genes encoding transcription factors have been shown to be central to the development of the anterior eye. Yet, these transcription factors and/or cofactors need to coordinate an incredibly complex array of interactions and communications between the tissues comprising the anterior eye. One most extensively studied gene, Pax6 (paired box homeotic gene 6) is expressed in all cell types of anterior eye lineage and clearly central to the timing and coordination of events during the formation of the anterior eye.3,5–10 Additionally, in recent years, substantial effort has been made in a search for other crucial factors of anterior eye development.11–15 These studies significantly advanced our knowledge on the progression of anterior eye diseases, such as squamous metaplasia, as well as on the differentiation of ocular surface ectoderm. However, it is still most clear that further identification of additional elements that are essential for providing tight and precise regulation during eye development will ensure our best chance to develop the most effective therapies for such devastating eye diseases. Pinin (Pnn/DRS/memA), a 140 kDa phosphoprotein associated with and localized in the nuclear speckles, has been shown to participate in the transcriptional regulation and RNA processing through its physical interaction with many transcriptional and splicing factors.16–26 We have previously shown that Pnn is essential for sustaining cell–cell adhesion and maintaining epithelia. The expression of exogenous Pnn in transformed cells drove these cells to the epithelial phenotype and reversed anchorage independent growth,23,24 while interfering with Pnn (eg, siRNA) in epithelial cell cultures caused the cells to disassociate and change growth qualities.18 Interestingly, Pnn hypomorphic mice (that exhibit general 90% knockdown in Pnn, resulting from inclusion of neo-cassette) displayed perinatal mortality with a wide range of developmental defects, including cardiac outflow tract and craniofacial malformation,19 and anterior eye dysgenesis (JHJ, unpublished data, 2007). Our most recent data indicate that Pnn plays a crucial role in the establishment and maintenance of epithelia throughout the body (JHJ, unpublished data, 2009). In vivo conditional inactivation of Pnn resulted in dramatic phenotypes in pulmonary and small intestinal epithelium, where viability and growth properties of the mutant epithelial cells were similar to controls, yet epithelial differentiation and morphogenetic programs were dramatically altered. These studies revealed Pnn to be involved with the maintenance of the epithelial differentiation and the specific epithelial phenotype. Here we report the Pax6-Cre (Le-Cre)–mediated deletion of Pnn in mice. The specific loss of Pnn in the ocular surface ectoderm resulted in severe malformation of lens placode-derived tissues including cornea and lens. Most interestingly, deletion of Pnn in the corneal epithelium resulted in the loss of corneal epithelial identity. The mutant corneal epithelia appeared epidermis-like, downregulated corneal keratins (K12), and turned on epidermal keratins (K10 and K14). This metaplasia of Pnn mutant corneal epithelium closely correlated with elevated β-catenin activity and misregulated p68 levels. Together, these data indicate that Pnn is essential for modulating and/or orchestrating the activities of major developmental factors of anterior eye segments.