Project Summary The liver is an essential organ and the only solid organ capable of regeneration. Despite this, liver regeneration is compromised in patients with advanced liver disease, and so understanding the mechanisms of liver regeneration has significant implications for regenerative medicine. Zebrafish have emerged as a powerful experimental system for studying regeneration, and there are several models of liver regeneration in zebrafish. I propose to thoroughly characterize the process of liver regeneration in zebrafish using a combination of classical tools and new techniques. I will use single-cell sequencing to identify more markers for described cell types, discover rare or transient cell populations, reconstruct lineage transitions, and elucidate the transcriptional responses to injury. I will validate these data by multicolor fluorescent in situ hybridization to gain spatial insight into gene expression. I will utilize a new technique to combine single-cell sequencing data with genetic lineage tracing to reveal the cellular basis of liver regeneration after severe liver injury. These findings will be validated with classical lineage tracing techniques in order to study the signaling pathways required for liver progenitor cells during regeneration. These data will further our understanding of the molecular and cellular basis of liver regeneration in zebrafish, aiding our ability to develop therapies to enhance liver regeneration in humans.