Comprehensive single cell mRNA profiling reveals a detailed roadmap for pancreatic endocrinogenesis

Deciphering mechanisms of endocrine cell induction, specification and lineage allocation <i>in vivo</i> will provide valuable insights into how the islets of Langerhans are generated. Currently, it is ill defined how endocrine progenitors segregate into different endocrine subtypes during development. Here, we generated a novel neurogenin 3 (Ngn3)-Venus fusion (NVF) reporter mouse line, that closely mirrors the transient endogenous Ngn3 protein expression. To define an <i>in vivo</i> roadmap of endocrinogenesis, we performed single cell RNA sequencing of 36,351 pancreatic epithelial and NVF⁺ cells during secondary transition. This allowed <i>Ngn3</i>^low endocrine progenitors, <i>Ngn3</i>^high endocrine precursors, <i>Fev⁺</i> endocrine lineage and hormone⁺ endocrine subtypes to be distinguished and time-resolved, and molecular programs during the step-wise lineage restriction steps to be delineated. Strikingly, we identified 58 novel signature genes that show the same transient expression dynamics as <i>Ngn3</i> in the 7260 profiled <i>Ngn3</i>-expressing cells. The differential expression of these genes in endocrine precursors associated with their cell-fate allocation towards distinct endocrine cell types. Thus, the generation of an accurately regulated NVF reporter allowed us to temporally resolve endocrine lineage development to provide a fine-grained single cell molecular profile of endocrinogenesis <i>in vivo</i>.