Date of Award
mammalian epidermis, tissue morphogenesis, skin apoptosis, alpha catenin, epithelium
The development and maintenance of epithelial tissues is regulated by a complex array of signal cues from adjacent cells, the extracellular milieu and intracellular signaling cascades. In the mammalian epidermis, these cues instruct the specification and invagination of hair follicles as well as the stratification and turnover of the interfollicular epidermis. These processes rely on a coordinate balance of tissue growth, differentiation and regulation of cell-cell adhesion to maintain the integrity of the epithelium. Understanding the interplay between the various pathways controlling tissue development requires model systems that recapitulate the events that occur in vivo. Genetic studies in Drosophila and other lower eukaryotes have uncovered many of the genes and pathways involved. However, genetic studies in mammals, where the relation to human development and disease is more direct are often hampered by the length of time required to generate mutations in the gene of interest. The ability to probe genetic interactions is even more limited in this system as the generation of double or triple mutants is even more inefficient. To circumvent these limitations, I have developed and optimized a lentivirus-based strategy to achieve rapid manipulation of the mammalian epidermis in vivo. Using ultrasound-guided in utero injections of fluorescently-traceable lentivirus particles carrying shRNA or Cre-recombinase into mouse embryos, I demonstrated a highly efficient, non-invasive, selective transduction of surface epithelium. Epidermal progenitors stably incorporated and propagated the desired genetic alterations. Importantly, I achieved epidermal-specific infection using small generic promoters from existing shRNA libraries, thus enabling rapid assessment of gene function and complex genetic interactions in skin morphogenesis and disease in vivo. Using this technology, I have developed a new quantitative method to ascertain whether a gene confers a growth advantage or disadvantage by measuring relative growth of mutant clones in a mosaic tissue. Taking the adherens junction protein Î±-catenin as a paradigm, this approach was used to uncover new insights into its role as a widely expressed tumor suppressor and regulator of epithelial integrity. Using simultaneous gene depletion I uncovered physiological interactions between Î±-catenin and the Ras- MAPK and Trp53 pathways in regulating skin proliferation and apoptosis, respectively. Surprisingly, the apoptotic cells were primarily localized in the suprabasal cells. I found that cells lacking Î±-catenin showed elevated focal adhesion signaling, and using the lentiviral knockdown approach, I found that co-depletion of focal adhesion signaling components reduced the protection from apoptosis afforded to the basal layer cells in the absence of Î±-catenin. These studies illustrate the strategy, its broad applicability for investigations of tissue morphogenesis, lineage specification and cancers, and yield new insights into the complex mechanisms of growth regulation in tissues.
Livshits, Geulah, "Rapid Functional Dissection of Genetic Networks Via RNAi In Mouse Embryos" (2011). Student Theses and Dissertations. 135.