Project Summary/Abstract Adult animals capable of whole-body regeneration are tasked with faithfully replacing any missing cell type, including the myriad of cell types within the nervous system. Most knowledge regarding the patterning and specification of neuronal populations comes from work focusing on embryonic development; however, limited work has been performed to identify mechanisms underlying the regeneration or re-specification of entire nervous systems within adult animals. The overall objective of this project is to uncover the cellular and molecular mechanisms that underlie neuronal cell specification, differentiation, and replacement within the context of an adult animal, using the highly regenerative acoel research model, Hofstenia miamia. Hofstenia has an organized nervous system composed of different neural cell types that it can regenerate fully because of a population of pluripotent adult stem cells called ‘neoblasts’. Notably, Hofstenia is amenable to mechanistic studies of regeneration and presents several advantages over more well-established invertebrate regeneration models, including accessible embryos that have enabled CRISPR/Cas9-based genome editing and stable transgenesis. This project will ask two major questions about nervous system regeneration within the adult nervous system: 1) What are the molecular/genetic regulators of neural cell type diversity during regeneration? 2) What are the cellular sources and dynamics that underlie differentiation of neural populations during regeneration? Single-cell RNAseq will identify candidate regulators of neural cell type diversity, focusing on transcription factors and receptors, during regeneration. Our preliminary scRNAseq data allows us to hypothesize sox4, vax1, and nkx2.4 homologs as important regulators of neural differentiation. Systemic RNAi in combination with microscopy in adults will identify the molecular mechanisms governing neural cell type identity during regeneration. In parallel, we plan to utilize a transgenic labeling technique to identify neural stem cell populations, determining their dynamics and contributions to differentiated neural populations during regeneration. These two questions allow us to test the hypothesis that a single neural stem cell population subfunctionalizes to form progenitor subtypes within Hofstenia. Cell and molecular mechanisms discovered in the regeneration of the adult nervous system in this work have the potential to inform human regenerative medicine with regards to neurodegenerative disease. My goal for the F31 is to equip myself with the computational, genetic, and theoretical skills necessary for a lifetime career in developmental biology to uncover the intricacies associated with animal regeneration. The Department of Organismic and Evolutionary Biology at Harvard University is a premier institution for this training.