Biological resilience is the ability of organisms to rebound from various stresses including tissue injury and exposure to toxins that cause DNA damage. Stem cells offer a potentially powerful line of defense against these insults through their ability to restore tissues, yet stem cells themselves are susceptible to deterioration from overuse or age, as a result of recurring cell divisions. To understand how stem cells can resist these types of insults, my lab studies one of the most resilient animals known, freshwater planarians. Planarians are famous for their ability to regenerate entire animals from tiny tissue fragments, due to an abundant population of stem cells. These stem cells appear to be inexhaustible: not only can they differentiate into any cell type, but they also maintain animal integrity throughout thousands of generations of asexual reproduction. Here, we will leverage the unique biology of planarian stem cells to identify conserved mechanisms responsible for their ability to regenerate repeatedly and to overcome significant genotoxic stress. The proposed experiments integrate prior findings with innovative tool development to advance our understanding of stem cell biology in this ideal invertebrate animal model. In the next five years, our work will occur in three primary research directions: (1) To regenerate entire animals, stem cell differentiation must be precisely coordinated. We recently identified a new signaling pathway involving the Roundabout receptor and Anosmin-1 that instructs stem cell differentiation to a specific body region. We will characterize the biochemistry and cell biology of this pathway to determine how stem cell activity is sculpted during regeneration. (2) Ionizing radiation causes DNA damage that is lethal to stem cells. We discovered two strategies that enable stem cells to overcome what was previously thought to be certain death. In the process, we uncovered key molecular differences in DNA damage response pathways in planarians that may reveal new strategies for DNA repair in less resilient animals. We will use molecular tools combined with genetic manipulations to mark sites of DNA damage and elucidate fundamental aspects of DNA repair, cell cycle progression, and apoptosis that enable planarians to withstand and recover from high doses of ionizing radiation. (3) To fully understand molecular and cellular aspects of regeneration, we need the ability to modify the genome modification, a technique that has not yet been developed in planarians. Building on preliminary findings, we will advance this technology using a stepwise approach, enabling the development of tools that can be applied across many studies. Together, these three lines of investigation will illuminate mechanisms that equip planarian stem cells with extreme plasticity and persistence throughout many thousands of divisions. Our work will inform strategies to mitigate stem cell exhaustion or enhance regeneration in other animals.