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.

Summary RNA-binding proteins (RBPs) and long non-coding RNAs (lncRNAs) are key regulators of gene expression, but their joint functions in coordinating cell fate decisions are poorly understood. Here we show that the expression and activity of the RBP TDP-43 and the long isoform of the lncRNA Neat1 , the scaffold of the nuclear compartment “paraspeckles,” are reciprocal in pluripotent and differentiated cells because of their cross-regulation. In pluripotent cells, TDP-43 represses the formation of paraspeckles by enhancing the polyadenylated short isoform of Neat1 . TDP-43 also promotes pluripotency by regulating alternative polyadenylation of transcripts encoding pluripotency factors, including Sox2 , which partially protects its 3′ UTR from miR-21 -mediated degradation. Conversely, paraspeckles sequester TDP-43 and other RBPs from mRNAs and promote exit from pluripotency and embryonic patterning in the mouse. We demonstrate that cross-regulation between TDP-43 and Neat1 is essential for their efficient regulation of a broad network of genes and, therefore, of pluripotency and differentiation.

Insulin and insulin-like growth factor 1 (Igf1) resistance in pancreatic β-cells causes overt diabetes, thus, therapeutic improvement may protect from β-cell failure 1-3 . Here, we identified a novel inhibitor of insulin (Insr) and Igf1 receptor (Igf1r) signalling in β-cells, which we named insulin inhibitory receptor (Inceptor; Iir). Inceptor contains an extracellular cysteine-rich domain with similarities to the Insr and Igf1r 4 and a mannose-6-phosphate domain found in the Igf2r 5 . Inceptor knock-out (KO) mice die within the first hours after birth with signs of hyperinsulinemia and hypoglycaemia. Molecular and cellular analysis of the Iir -/embryonic and postnatal pancreas showed increased Insr/Igf1r activation, resulting in augmented β-cell proliferation and mass. Similarly, inducible β-cellspecific Iir -/-KO in adult mice and in ex vivo islets led to increased Insr/Igf1r activation and β-cell proliferation, resulting in improved glucose tolerance in vivo. Mechanistically, Inceptor interacts with Insr and Igf1r to facilitate clathrinmediated endocytosis for receptor desensitisation. Blocking this physical interaction using monoclonal antibodies against the extracellular domain of Inceptor retained Inceptor and Insr at the plasma membrane to sustain Insr/Igf1r activation in β-cells. Taken together, Inceptor shields insulin-producing β-cells from constitutive pathway activation and provides a molecular target for Insr/Igf1r sensitisation and potential diabetes therapy.