Differential nuclear expression of Yap in basal epithelial cells across the cornea and substrates of differing stiffness

Abstract

Corneal epithelium is maintained throughout life by well-orchestrated proliferation of limbal epithelial stem cells, followed by migration and maturation centripetally across the ocular surface. The present study sets out to explore the role tissue stiffness (compliance) may have in directing both differentiation and centripetal migration of limbal epithelial stem cells during homeostasis. For that, we analysed the localization of the Yes-associated protein (Yap), a transcriptional co-activator previously shown to mediate cellular response and mechanical stimuli. Using both models of ocular surface compliance and normal bovine corneas we evaluated the nuclear/cytoplasmic expression ratio of Yap. Expression levels within corneal epithelial cells were compared in situ between the limbus and central cornea, and in vitro between limbal epithelial stem cells expanded upon biomimetic collagen gels of increasing stiffness. Nuclear expression of Yap was shown to increase within the expanded cells upon substrates of increasing stiffness. Subsequently, Yap was used as a novel molecular probe to investigate the mechanical microenvironment within a normal ocular surface. The in situ localization of Yap was predominantly cytoplasmic within basal limbal epithelial cells and nuclear within basal central corneal epithelial cells. Furthermore, nuclear p63 expression was not co-localized with Yap in basal limbal epithelial cells. In conclusion, the current investigation provides new insights into the relationship between Yap and distinct cell populations across the ocular surface indicating that cells experience a different mechanical environment between the limbus and central cornea. A new hypothesis is put forward, in which centripetal differences in substrate stiffness drives the migration and differentiation of limbal epithelial stem cells, thus controlling corneal epithelium homeostasis.