Project Summary The capacity to regulate stem and progenitor cells for regeneration is widespread in the animal kingdom and has attracted investigation for centuries. Evolution has selected for mechanisms involved in wound repair and tissue regrowth that are the dreams of regenerative medicine. Uncovering the principles of regeneration in case study organisms should identify processes that naturally promote or limit regeneration, enabling future development of therapeutic approaches to tissue damage repair. Planarians are flatworms capable of regenerating any missing body part, including new heads. Their regenerative powers have combined with ease of experimentation to make them a classic regeneration model. Planarian regeneration involves adult stem cells called neoblasts, which we previously showed can display pluripotency at the single cell level (cNeoblasts). A major direction of our research addresses how fate specification occurs in regeneration. Extensive work indicates that fate choices are made within the neoblasts (called specialized neoblasts), with the regeneration outgrowth at the wound (a blastema) being a composite of different fate-specified cells. This highlights the step of fate-choice in neoblast stem cells as central for understanding the mechanistic basis for planarian regeneration. We seek to understand "specificity" in regeneration, in which diverse injuries appear to result in responses tailored to the identity of missing tissue. We aim to distinguish between the possible existence of surveillance systems indicating the presence or absence of differentiated cell types and what we named target-blind regeneration. In target-blind regeneration, progenitor production occurs at a low basal rate sufficient for repair from small wounds, without needing tissue surveillance; we hypothesize tissue-specific progenitor production is primarily regulated by wound-induced proliferation combined with positional information. We aim to understand how positional information regulates stem cell fate choice during tissue turnover, and dynamically during regeneration. Our prior work on a planarian whole-animal cell-type transcriptome atlas indicates the existence of over 100 adult cell types. We seek to understand what processes within neoblast stem cells regulate how they make any one of so many possible choices. We will study the pattern of fate choices using spatial transcriptomics and seek to distinguish between a highly regulated fate- specification process, such as by extrinsic local tissue cues, and a more stochastic process internal to the stem cells. Finally, we will investigate how adult progenitors generated from stem cells bring about the restoration of tissue architecture in regeneration. Our prior work indicates that migratory targeting by extrinsic cues combines with self-organization of progenitors with their target tissue to generate and maintain tissue pattern in regeneration. We will study the molecular bases for these proces...