Whole-Mount Assays for Gene Induction and Barrier Formation in the Developing Epidermis
The skin as a surface organ is uniquely accessible for whole embryo/fetal analyzes of developmental changes, such as gene induction, protein expression, formation of epidermal-derived appendages such as hair follicles, and formation of the protective barrier. Such analyses have emphasized the heterogenous nature of skin development, perhaps not surprising because epidermal development is programmed by heterogenous underlying mesenchyme. It is necessary to account for this heterogeneity by precisely matching body sites when correlating sequential events during development, for example, the activation of gene expression. In this chapter, protocols designed to assay whole-mount in situ hybridization and whole-mount barrier formation are presented. Formation of the protective barrier is the endpoint of epidermal terminal differentiation and defects in this process are reflected in failure, acceleration, or delay in barrier formation. Hence, these latter assays are of particular value as a rapid initial assay for epidermal developmental defects in genetically modified organisms.
Key WordsWhole-mount in situ hybridization gene induction dye-penetration dye-exclusion barrier assays
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