Massive single-cell mRNA profiling reveals a detailed roadmap for pancreatic endocrinogenesis

Abstract

Deciphering mechanisms of endocrine cell induction, specification and lineage allocation in vivo 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 Neurogenin3 (Ngn3)-Venus fusion (NVF) reporter mouse line, that closely mirrors the transient endogenous Ngn3 protein expression. To define an in vivo roadmap of endocrinogenesis, we performed single-cell RNA-sequencing of 36,351 pancreatic epithelial and NVF+ cells during secondary transition. This allowed to time-resolve and distinguish Ngn3low endocrine progenitors, Ngn3high endocrine precursors, Fev+ endocrine lineage and hormone+ endocrine subtypes and delineate molecular programs during the stepwise lineage restriction steps. Strikingly, we identified 58 novel signature genes that show the same transient expression dynamics as Ngn3 in the 7,260 profiled Ngn3-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 in vivo.