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 neurogenin 3 (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 Ngn3 low endocrine progenitors, Ngn3 high endocrine precursors, Fev + endocrine lineage and hormone + endocrine subtypes to be distinguished and timeresolved, 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 Ngn3 in the 7260 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.

Single cell genomics has become a popular approach to uncover the cellular heterogeneity of progenitor and terminally differentiated cell types with great precision. This approach can also delineate lineage hierarchies and identify molecular programmes of cell-fate acquisition and segregation. Nowadays, tens of thousands of cells are routinely sequenced in single cell-based methods and even more are expected to be analysed in the future. However, interpretation of the resulting data is challenging and requires computational models at multiple levels of abstraction. In contrast to other applications of single cell sequencing, where clustering approaches dominate, developmental systems are generally modelled using continuous structures, trajectories and trees. These trajectory models carry the promise of elucidating mechanisms of development, disease and stimulation response at very high molecular resolution. However, their reliable analysis and biological interpretation requires an understanding of their underlying assumptions and limitations. Here, we review the basic concepts of such computational approaches and discuss the characteristics of developmental processes that can be learnt from trajectory models.

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Dedifferentiation of insulin-secreting β cells in the islets of Langerhans has been proposed to be a major mechanism of β-cell dysfunction. Whether dedifferentiated β cells can be targeted by pharmacological intervention for diabetes remission, and ways in which this could be accomplished, are unknown as yet. Here we report the use of streptozotocin-induced diabetes to study β-cell dedifferentiation in mice. Single-cell RNA sequencing (scRNA-seq) of islets identified markers and pathways associated with β-cell dedifferentiation and dysfunction. Single and combinatorial pharmacology further show that insulin treatment triggers insulin receptor pathway activation in β cells and restores maturation and function for diabetes remission. Additional β-cell selective delivery of oestrogen by Glucagon-like peptide-1 (GLP-1-oestrogen conjugate) decreases daily insulin requirements by 60%, triggers oestrogen-specific activation of the endoplasmic-reticulum-associated protein degradation system, and further increases β-cell survival and regeneration. GLP-1-oestrogen also protects human β cells against cytokineinduced dysfunction. This study not only describes mechanisms of β-cell dedifferentiation and regeneration, but also reveals pharmacological entry points to target dedifferentiated β cells for diabetes remission.There are amendments to this paper NATURE METABoLiSM | VOL 2 | FEBRUARy 2020 | 192-209 | www.nature.com/natmetab 192 Articles NATuRE METAboLiSM autoimmunity in the mSTZ model permits the investigation of the fate of those remaining β cells and the effect of pharmacological treatment on β-cell protection and regeneration.