The trident of the pancreatic duct : stemness, morphogenesis and endocrinogenesis

Abstract: Different subtypes of diabetes are all featured by the deterioration and loss of functional β-cell. Currently, there is still lack of therapeutic strategies to cure diabetes. However, harnessing the innate stem cell population to become functional β-cell could be a potential way for future treatment. Mammalian animals, including mouse and human, can barely replenish the lost β-cell in the disease state; however, early vertebrates, especially zebrafish, has astonishing tissue regeneration capacity. In particular, zebrafish can quickly recover from the extreme β-cell loss and recovery body glucose level very quickly. Understanding the underlying cellular and molecular events is pivotal for the drug discovery in translational studies to treat diabetes. In Paper I, we introduced a compound, named CID661578, which we identified from a large- scale chemical screening experiment. Combining yeast hybrid assay, biochemistry experiments, polysome sequencing, single-cell RNA-seq and genetic zebrafish mutant, we confirmed that mknk2b in zebrafish and MNK2 in human are the major functional target. Functional testing indicated that CID661578 can induce duct-to-β-cell neogenesis in both zebrafish and neonatal pig islet. In Paper II, we introduced a novel CRISPR/Cas9 knock-in method to generate zebrafish lines for multiple utility (i.e. cell labeling and lineage tracing). We creatively used double-stranded DNA with 5’ modification as the donor. Such clone-free, one-step knock-in strategy allow researchers to target different loci in a quick and scalable fashion. Using the newly generated lines, we are able to delineate the developmental paths of zebrafish pancreas and liver and explore the origins of the regenerative hepatocytes under different injury conditions. In Paper III, the newly developed knock-in tools allow us to decipher the developmental paths of zebrafish endocrinogenesis in normal and β-cell ablation conditions. Combining single-cell RNA-seq, lineage tracing, cell targeted ablation, immunofluorescence, in situ hybridization, we identified a previously unrecognized ductal heterogeneity in zebrafish pancreas as well as the endocrine precursor cells during β-cell development. We delineate the key cellular and molecular events in β-cell differentiation and de-differentiation and pinpoint the distinct origins of β-cells during the development and regenerative conditions. This study offers a good resource and provides novel mechanistic insights into β-cell development and regeneration in zebrafish.