Optical and Biochemical Dissection of Connexin and Disease-Linked Connexin Mutants in 3D Organotypic

The epidermis is a complex tissue composed principally of differentiated keratinocytes that form a keratinized stratified squamous epithelium. The gap junction proteins, connexins (Cx), are differentially expressed throughout the stratified layers of the epidermis and their exquisite regulation appears to govern the delicate balance between cell proliferation and differentiation in normal skin homeostasis and in wound healing. In the last 10 years, germ line mutations in the genes encoding five connexin family members have been linked to various types of skin diseases that appear to offset the balance between keratinocyte differentiation and proliferation. Consequently, in order to determine how these connexin gene mutations manifest as skin disease, disease-linked mutants must be expressed in 3D organotypic epidermis reference models that attempt to mimic the human condition. Given the complexity of organotypic epidermis, confocal optical and biochemical dissection of connexin or disease-linked connexin mutants within the regenerated epidermal layer is required. The procedures necessary to assess the architectural characteristics of genetically modified organotypic epidermis and its state of differentiation will be described in this chapter.