Assessing the In Vivo Epidermal Barrier in Mice: Dye Penetration Assays

Abstract

INTRODUCTION The formation of a properly functioning epidermal barrier is a prerequisite for terrestrial life. The epidermis not only protects from external influences such as pathogens, chemicals, and UV light, but also prevents dehydration. To maintain proper barrier function, the epidermis undergoes a dynamic turnover driven by proliferating keratinocytes in the basal layer that, upon induction of differentiation, will move upward through the stratum spinosum and stratum granulosum while undergoing a terminal differentiation process to ultimately form the stratum corneum (SC). This layer is in direct contact with the outside environment and provides important structural and innate immune barrier properties. Barrier function is initiated when stratum granulosum keratinocytes start secreting and crosslinking specific proteins and lipids into the intercellular space and at the same time transform their membrane into a so-called cornified envelope (e.g., by linking different structural proteins such as loricrin to the inner side of the cell membrane). Consequently, keratinocytes shed their nucleus to become corneocytes forming the SC. In recent years it has become clear that the SC is not the sole structure providing barrier properties to the epidermis. It is now clear that specialized intercellular junctions, called tight junctions (TJs), are present between cells of the granular layer, where they ensure proper barrier function (Furuse et al., 2002; Tunggal et al., 2005). TJs have been well characterized in simple epithelia where they form a paracellular sizeand ion-specific barrier to separate tissue compartments. The size and ion selectivity of TJs is mainly based on which members of the claudin family of transmembrane proteins are located in the TJs of different tissues. The epidermis expresses several claudins, and loss of claudin-1 in mice results in perinatal lethality attributable to rapid dehydration (Furuse et al., 2002). In addition, TJs are crucial for immune surveillance by Langerhans cells (Kubo et al., 2009). The importance of the two barriers is further confirmed by observations that mutations in SC proteins such as filaggrin are linked to diseases characterized by an impaired skin barrier, such as atopic dermatitis and ichthyosis vulgaris (Palmer et al., 2006; Smith et al., 2006). In addition, human claudin-1 mutations are associated with neonatal sclerosing cholangitis associated with ichthyosis (Hadj-Rabia et al., 2004). However, the exact molecular mechanisms of how TJs and the SC contribute to the formation, maintenance, and restoration of the skin barrier are not well understood. In addition, it is not known whether and how TJs and the SC cooperate to form a fully functional skin barrier. Transgenic mouse models have been helpful tools to investigate how proteins implicated in epidermal barrier function contribute to the formation and maintenance of skin barrier function. Next to assessing whether such mice have a BENEFITS OF DYE PENETRATION ASSAYS