R35 GRANT ABSTRACT My lab is broadly interested in understanding how cellular differentiation is controlled within a continuously renewing epithelial tissue. Conserved features of these tissues that are not fully understood and unify studies across experimental systems include a flexible niche structure, a ?transit amplification? stage which typically has significant cellular plasticity, and the ability of neighboring stem cell lineages to compete for niche occupancy. To understand these emergent properties of tissues, we have focused on approaches that allow for the study of cell behaviors within the native, in vivo context in at cellular resolution. Our primary model system is the follicle epithelium of the Drosophila ovary, and we have recently extended our studies into the mouse intestinal epithelium. Our contributions over the past ten years include identifying the source and identity of the follicle stem cell (FSC) niche ligands, defining a self-renewal network for FSCs, describing new mechanisms that promote the segregation of stem cell and daughter cell fates, and the establishment and use of the FSC lineage as a model for understanding stem cell niche competition. Our current studies are investigating three interconnected areas. First, we created a cell atlas of the Drosophila ovary that describes the identity, position, and gene expression profile of over a dozen known and novel cell types. This project has provided useful new tools that are allowing us to investigate the lineage plasticity of cells in the tissue and has led, for example, to the discovery that niche cells can convert to stem cells during physiological stress. In addition, these tools provide us with a new opportunity to study how a dynamic population of niche cells is able to maintain a stable pool of FSCs amid changing tissue demands. Second, we are investigating the molecular mechanisms that govern stem cell niche competition. We have identified a broad class of alleles that cause hypercompetition for the niche, and we are using genetics, quantitative imaging, and mathematical modeling to understand the basis for selection of one lineage over another. We have also extended these studies into the mouse intestinal epithelium and found that the process is at least partially conserved. Third, we are investigating the role of intracellular pH (pHi) in regulating cell fate decisions. We demonstrated that pHi increases during differentiation in both the FSC lineage and mouse embryonic stem cells, and that this increase in pHi is necessary for differentiation. In unpublished studies, we discovered a similar requirement for increased pHi in the mouse intestinal stem cell lineage. Currently, we are focused on understanding how pHi regulates cell fate, with an emphasis on candidate ?pH sensor? proteins, such as ?-catenin, that have a pKa within the physiological range. For these proteins, the gain or loss of a proton functions like a post-translational modification, thus linking pH dynamics to changes in protein activities that may affect cell fate. By studying these emergent properties within well-characterized Drosophila and mouse epithelia, we are gaining detailed insights into the process of normal tissue homeostasis that will provide a foundation for a better understanding of how tissue homeostasis fails during aging and disease.
The proposed research will provide a foundation for understanding how cell fate decisions are controlled within an epithelial stem cell lineage. Epithelial stem cells are central to both normal homeostasis and the progression of diseases such as cancer, so understanding how they are regulated their native, in vivo contexts is important for the development of new diagnostics and therapeutics. The proposed project will also contribute to the NIH mission to foster fundamental creative discoveries by investigating new and poorly understood topics such as stem cell niche competition and the role of pH dynamics in cell fate decisions.