PROJECT SUMMARY ABSTRACT Cells within epithelial tissues are continually being eliminated by apoptosis and replaced by cell proliferation, however the mechanisms that coordinate cell removal with cell division to retain constant cell numbers remain unknown. Failure to coordinate the birth and death of cells can lead to dysregulation of population numbers and compromised barrier function, or conversely, tissue hyperplasia and carcinoma formation. Thus, a thorough understanding of the genetic underpinnings guiding cellular turnover in epithelial tissues will provide insight into molecular pathways that can be leveraged against diverse human pathologies by enhancing the removal and replacement of defective cells. The goal of this proposal is to define the cell and molecular mechanisms that regulate cell turnover in epithelial tissues to maintain appropriate overall population numbers. My recent results suggest that clearance of excess or defective cells was a major influence in whether neighboring cells would divide, extrude or die. Importantly, this work also suggested that alterations in the ability to rapidly clear apoptotic cells from the epithelial tissues may lead to several epithelial pathologies, including decreased barrier function in the intestinal epithelium or the accumulation of dangerous cells to promote carcinoma formation. Yet, a model system has been lacking to study how changes in apoptotic cell clearance could impact cell turnover and tissue maintenance in living epithelia. To investigate cell turnover in a living epithelial tissue, we have developed a toolset to perturb gene function and perform live imaging of division and death in the epithelia of the developing zebrafish, providing unparalleled access to analyze cell turnover in real time. Using the developing zebrafish to study cellular turnover in an epithelial bilayer, we have uncovered that induction of damage in a subset of basal epithelial cells promotes live cell neighbors to act as phagocytes that rapidly clear the apoptotic cellular debris. The basal stem cells then undergo division to compensate for the cell loss and maintain tissue integrity and function. Our preliminary data suggests that inhibition of either cell death or WNT signaling eliminates the apoptosis-induced division and results in failed regeneration. Further, genetic overexpression of WNT signaling in the context of a damage response led to an increase in overall cell numbers. In the following proposal, we will test the hypothesis that clearance of WNT-containing apoptotic cells by neighboring stem cells directly influences their proliferation to drive cell turnover in epithelia. In Aim 1, we will determine how removal of dying cells stimulates stem cell-mediated replacement. In Aim 2, we will define the molecular mechanisms guiding apoptosis-induced proliferation to maintain overall cell numbers. In Aim 3, we will determine if apoptotic bodies and microparticles can promote stem cell proliferation. Together, these studies will help reveal the cell and molecular events regulating epithelial tissue homeostasis and how alterations lead to human pathogenic conditions.

PROJECT NARRATIVE Altered epithelial homeostasis underlies several pathological conditions, from decreased turnover during barrier function disease to increased turnover and localized hyperproliferation to fuel carcinogenesis. The proposed studies will define how apoptotic cells influence their neighbors to induce proliferation to promote their own replacement and drive cell turnover. Discoveries arising from this work will improve our ability leverage endogenous mechanisms that to potentially treat multiple diseases that feature an epithelium with decreased form or function.