Successful regeneration of tissues requires transient increases in stem cell plasticity, proliferation, and differentiation, in order to produce new cells that integrate with preexisting tissues and organs. Pathways governing these critical behaviors have been identified, but how injury signals can trigger stem cell proliferation and differentiation of cells necessary for regeneration remains poorly understood. In most model organisms, regenerative capacity is limited and stem cells are scarce, which has made it difficult to pinpoint the mechanisms regulating stem cell proliferation and differentiation after injury. By contrast, the planarian flatworm Schmidtea mediterranea has abundant stem cells that are activated by injury and fuel continuous regeneration. Like embryonic stem cells, planarian stem cells have the capacity to differentiate into any type of tissue. These pluripotent stem cells can be readily identified, monitored, purified, and thoroughly profiled at the molecular level. We recently made two important discoveries that form the foundation of this proposal. First, injury of any type appears to protect stem cells from lethal radiation, because it halts the cell cycle and fewer stem cells undergo apoptosis. Second, we pioneered a chemical method to selectively remove a single organ, the pharynx. Pharynx regeneration requires the upregulation of the conserved Forkhead transcription factor FoxA in a discrete subset of stem cells immediately after this targeted injury. We find that the extracellular signal-regulated kinase (ERK) is a central driver of these behaviors. ERK promotes differentiation in cultured stem cells, but how it is activated after injury is poorly understood. Together, these findings establish our central hypothesis, which is that injury synchronizes the cell cycle, enabling local cues to channel stem cell differentiation toward discrete cell fates. In Aim 1, we will determine how injury induces cell cycle arrest in stem cells after radiation. We will examine DNA repair and test the function of conserved genes that are upregulated after injury. In Aim 2, we will dissect the mechanisms driving organ-specific regeneration by purification and single-cell sequencing of stem cells proliferating after organ loss. We will identify receptors enriched on these cells, and test their function in organ regeneration to determine if they act upstream of FoxA. In Aim 3, we will identify the upstream receptors that activate MAP kinase signaling in stem cells with combinations of RNAi, pharmacology and biochemistry. This proposal exploits our ability to challenge stem cells with precise insults, providing a lens into the mechanisms that enable flexible stem cell responses during injury and homeostasis. Understanding the molecular mechanisms that govern stem cell behavior in a physiologically-relevant context will inform the design of future strategies for regenerative medicine technologies.