Sequential progenitor states mark the generation of pancreatic endocrine lineages in mice and humans

The pancreatic islet contains multiple hormone+ endocrine lineages (alpha, beta, delta, PP and epsilon cells), but the developmental processes that underlie endocrinogenesis are poorly understood. Here, we generated novel mouse lines and combined them with various genetic tools to enrich all types of hormone+ cells for well-based deep single-cell RNA sequencing (scRNA-seq), and gene coexpression networks were extracted from the generated data for the optimization of high-throughput droplet-based scRNA-seq analyses. These analyses defined an entire endocrinogenesis pathway in which different states of endocrine progenitor (EP) cells sequentially differentiate into specific endocrine lineages in mice. Subpopulations of the EP cells at the final stage (EP4-early and EP4-late) show different potentials for distinct endocrine lineages. epsilon cells and an intermediate cell population were identified as distinct progenitors that independently generate both alpha and PP cells. Single-cell analyses were also performed to delineate the human pancreatic endocrinogenesis process. Although the developmental trajectory of pancreatic lineages is generally conserved between humans and mice, clear interspecies differences, including differences in the proportions of cell types and the regulatory networks associated with the differentiation of specific lineages, have been detected. Our findings support a model in which sequential transient progenitor cell states determine the differentiation of multiple cell lineages and provide a blueprint for directing the generation of pancreatic islets in vitro. Overall design: The overall goal of this study was to comprehensively delineate the developmental trajectory of pancreatic endocrine cells using single-cell RNA-seq (scRNA-seq) analyses. We performed scRNA-seq on EP cells and hormone+ cells from embryonic day (E) 13.5 to postnatal day (P) 3 mice using the Smart-seq2 method. To enrich low-abundance EP cells and their descendant endocrine lineages from the pancreas, we treated Ngn3-CreER;Rosa-RFP mice with a single tamoxifen pulse for 1 or 2 days, and collected RFP+ cells from E13.5-E18.5 by fluorescence-activated cell sorting (FACS). To enrich low-abundance differentiated e-, d-, and PP-cells, we generated fluorescent-labeled knock-in Ghrl-P2A-CFP, Sst-P2A-BFP and Ppy-P2A-mNeptune mouse lines. To obtain an unbiased and comprehensive distribution of EP cells and endocrine lineages, we generated 10x Genomics scRNA-seq datasets of E16.5, E17.5, and E18.5 pancreatic cells that were separately from the dorsal and ventral pancreas.