PROJECT SUMMARY During development, the formation of heterogenous tissues, organs, and cellular networks depends on a careful balance between cellular proliferation, quiescence, and differentiation. Once cells have fully differentiated, their ability to proliferate or differentiate are most often lost, and so most adult cells are generally incapable of self- regeneration or repair. The development of regenerative therapies will require harnessing this latent ability, but first we need a deeper understanding of the processes that control it. Thus, understanding the genetic architecture underlying quiescent progenitor behavior is key to developing and applying emerging therapies with new technologies such as cellular reprogramming or genome editing. The objective of this proposal is to characterize the regulation and functions of potentially important genes controlling quiescence, differentiation, and proliferation in marine invertebrate and chordate Ciona robusta. Ciona are among our closest invertebrate relatives, and so during development we share similar structural and molecular features. However, Ciona undergo a dramatic conversion from larval to adult forms called metamorphosis, when the larval body plan degenerates and is replaced by quiescent progenitors which must bypass programmed cell death to reemerge post-metamorphosis and generate the adult body plan. One such cell population are larval neural progenitors called Neck cells which are established in a discrete stem cell niche-like compartment. The signaling pathways and genetic components that direct Neck entry, maintenance, and exit from quiescence remain uncharacterized. I propose using the Neck cell population as a model to identify unique mechanisms regulating quiescence and regeneration that can be harnessed for future therapies. The rationale for this proposal is that, by exploiting the tractability of Ciona, the accessibility of these Neck cells, and their stereotyped cellular behaviors, I can closely examine regulatory control of Neck cell quiescence, proliferation, and differentiation. I will do so by pursuing two specific aims. 1) To investigate the control of Neck quiescence and proliferation during the larval stage by the integration of extracellular cues and intracellular transcriptional control. 2) To investigate a novel mechanism for transcriptional priming and delay of Neck cell differentiation. I will pursue these aims using an innovative approach that combines cell lineage-specific, CRISPR/Cas9-based somatic gene knockouts and fluorescence microscopy. The expected outcomes of the proposed work include identifying previously unrecognized functions for conserved but poorly studied genes in neurodevelopment and how their spatiotemporal regulation can be instructive for the precise timing of quiescence. This will establish a foundation for a targeted investigation of neurodevelopmental processes underlying a wide range of human disorders.