Project Summary/Abstract Diabetes is a growing global health problem that requires improved life-long treatments. A more universal approach to treat type one diabetes (T1D) would be inducing endogenous cells to restore lost cells. Therefore, my proposed work aims to discover the cellular and molecular mechanism driving -cell neogenesis to identify pathways that can be exploited to restore cells in diabetics. We use zebrafish as a model system because they have specialized ducal cells called centroacinar cells (CACs) that behave as -cell progenitors. All CACs are Notch responsive, but cells that have lower Notch activity are more likely to differentiate. All CACs express sox9b, and our lab has shown that sox9b haploinsufficiency in zebrafish accelerates -cell regeneration. To better understand the progenitor behavior of CACs, our lab used bulk RNA-seq to uncover the CAC transcriptome. One major issue with bulk RNA-seq is that it masks CAC heterogeneity and cannot be used to reveal cellular and molecular mechanisms driving CAC differentiation at the single-cell level. I propose to better understand the cellular and molecular mechanisms of CACs by addressing two major aims: (1) Characterize cellular heterogeneity within the CAC population using single-cell RNA-seq and (2) Investigate transcriptome changes as CACs transition into cells during regeneration. (1) To characterize the variation in the CAC population, CACs will be isolated from wild type (WT) adult zebrafish pancreata using fluorescent activated cell sorting (FACS). Isolated CACs will be subject to scRNA-seq and cluster analysis will be applied to separate the CAC transcriptomes into distinct subpopulations. (2) I will use a transgenic zebrafish model to induce -cell ablation. At a halfway-point during regeneration, I will isolate CACs by FACS and will use these CACs for scRNA-seq. Cluster analysis and pseudotime analysis will be applied to order CACs and identify a mechanism for regeneration. For both aims, I will perform appropriate in situ experiments to visualize CAC subpopulations and confirm whether ‘new’ genes identified from my pseudotime model are associated with -cell differentiation. My project will elucidate the natural variation that exists within the CAC population and will uncover molecular mechanisms driving -cell regeneration. My lab and others will be able to use my validated data to gain a better understanding of the potential therapeutic routes that can be exploited to improve the treatment of T1D patients.