Elucidating cellular competition in mammalian epithelium and the role of Myh9.

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I plan to establish a research program that will stand out for taking a comprehensive and quantitative approach to functional studies in epithelial biology in its native, physiological environment. My goal is to identify molecular and cellular mechanisms that regulate epithelial tissue growth in adult homeostasis, cancer and chronic as well as cancer-associated inflammation. For the NSERC Discovery Grant project, I am proposing to elucidate the intimate relationship between cellular competition of tissue resident stem cells, cytoskeletal components such as myosin IIa and DNA damage repair. In addition, we will establish and refine our in vivo CRISPR genome-editing capabilities to genetically manipulate an intact epithelium during development and tissue homeostasis.***This project centers around one of the fundamental questions in biology, namely how stem cells balance self-renewal, differentiation and tissue homeostasis. As adult stem cells are a life-time reservoir for various lineage-specific cells, they are especially sensitive to spontaneous DNA damage. Mutations that impinge on their self-renewal or differentiation capacity may lead to tissue atrophy (organ degeneration) and accelerate aging due to stem cell exhaustion, while mutations resulting in a proliferative advantage can result clonal expansion and even cell competition. Central to the DNA damage response is the p53 pathway, which acts as relay to sense the damage and either triggers cell cycle arrest for DNA repair to happen or triggers cell death. Interestingly, we recently identified myosin IIa, an integral part of the actin-cytoskeleton, to be absolutely required for DDR-induced p53 activation however; the exact molecular mechanism remains elusive. We therefore plan to perform a panel of biochemical and proteomics experiments to elucidate the exact molecular machinery involved in myosinIIa-p53 interaction. Additionally, we will build upon our unique in vivo RNAi injection methodology and establish novel direct in vivo CRISPR genome-editing capabilities to study myosin IIa in stem cell behavior and cellular competition during development and adult homeostasis using mouse skin epidermis as our model system. Using this technology, we will then develop a Systems Biology screening approach, which will allow us to screen for and characterize essential factors governing cell competition for the first time in a mammalian organ system.***Importantly, our in vivo approach circumvents all limitations of conventional cell culture screens and assesses gene function in cells embedded within physiological tissue environment of immune-competent mice, which is paramount esp. for stem and progenitor cells, which heavily depend on stromal cues and niche signals. The technical expertise gained from those studies will certainly also be leveraged in our other projects and will be the basis for a rich collaborative platform. **