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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 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 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 ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Human embryonic stem cells (hESCs) are a potential unlimited source of insulin-producing β cells for diabetes treatment. A greater understanding of how β cells form during embryonic development will improve current hESC differentiation protocols. All pancreatic endocrine cells, including β cells, are derived from Neurog3-expressing endocrine progenitors. This study characterizes the single-cell transcriptomes of 6,905 mouse embryonic day (E) 15.5 and 6,626 E18.5 pancreatic cells isolated from Neurog3-Cre; Rosa26 mT/mG embryos, allowing for enrichment of endocrine progenitors (yellow; tdTomato + EGFP) and endocrine cells (green; EGFP). Using a NEUROG3-2A-eGFP CyT49 hESC reporter line (N5-5), 4,462 hESC-derived GFP+ cells were sequenced. Differential expression analysis revealed enrichment of markers that are consistent with progenitor, endocrine, or previously undescribed cell-state populations. This study characterizes the single-cell transcriptomes of mouse and hESC-derived endocrine progenitors and serves as a resource (https://lynnlab.shinyapps.io/embryonic_pancreas) for improving the formation of functional β-like cells from hESCs., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mutations in Hes1 , a target gene of the Notch signalling pathway, lead to ectopic pancreas by a poorly described mechanism. Here, we use genetic inactivation of Hes1 combined with lineage tracing and live imaging to reveal an endodermal requirement for Hes1, and show that ectopic pancreas tissue is derived from the dorsal pancreas primordium. RNA-seq analysis of sorted E10.5 Hes1 +/+ and Hes1 -/- Pdx1-GFP + cells suggested that upregulation of endocrine lineage genes in Hes1 -/- embryos was the major defect and, accordingly, early pancreas morphogenesis was normalized, and the ectopic pancreas phenotype suppressed, in Hes1 -/- Neurog3 -/- embryos. In Mib1 mutants, we found a near total depletion of dorsal progenitors, which was replaced by an anterior Gcg + extension. Together, our results demonstrate that aberrant morphogenesis is the cause of ectopic pancreas and that a part of the endocrine differentiation program is mechanistically involved in the dysgenesis. Our results suggest that the ratio of endocrine lineage to progenitor cells is important for morphogenesis and that a strong endocrinogenic phenotype without complete progenitor depletion, as seen in Hes1 mutants, provokes an extreme dysgenesis that causes ectopic pancreas., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Direct lineage reprogramming can convert readily available cells in the body into desired cell types for cell replacement therapy. This is usually achieved through forced activation or repression of lineage-defining factors or pathways. In particular, reprogramming toward the pancreatic β cell fate has been of great interest in the search for new diabetes therapies. It has been suggested that cells from various endodermal lineages can be converted to β-like cells. However, it is unclear how closely induced cells resemble endogenous pancreatic β cells and whether different cell types have the same reprogramming potential. Here, we report in vivo reprogramming of pancreatic ductal cells through intra-ductal delivery of an adenoviral vector expressing the transcription factors Pdx1, Neurog3, and Mafa. Induced β-like cells are mono-hormonal, express genes essential for β cell function, and correct hyperglycemia in both chemically and genetically induced diabetes models. Compared with intrahepatic ducts and hepatocytes treated with the same vector, pancreatic ducts demonstrated more rapid activation of β cell transcripts and repression of donor cell markers. This approach could be readily adapted to humans through a commonly performed procedure, endoscopic retrograde cholangiopancreatography (ERCP), and provides potential for cell replacement therapy in type 1 diabetes patients., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The production of insulin-producing β cells from human embryonic stem cells (hESCs) in vitro represents a promising strategy for a cell-based therapy for type 1 diabetes mellitus. To explore the cellular heterogeneity and temporal progression of endocrine progenitors and their progeny, we performed single-cell qPCR on more than 500 cells across several stages of in vitro differentiation of hESCs and compared them with human islets. We reveal distinct subpopulations along the endocrine differentiation path and an early lineage bifurcation toward either polyhormonal cells or β-like cells. We uncover several similarities and differences with mouse development and reveal that cells can take multiple paths to the same differentiation state, a principle that could be relevant to other systems. Notably, activation of the key β-cell transcription factor NKX6.1 can be initiated before or after endocrine commitment. The single-cell temporal resolution we provide can be used to improve the production of functional β cells., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The current model for endocrine cell specification in the pancreas invokes high-level production of the transcription factor Neurogenin 3 (Neurog3) in Sox9+ bipotent epithelial cells as the trigger for endocrine commitment, cell cycle exit, and rapid delamination toward proto-islet clusters. This model posits a transient Neurog3 expression state and short epithelial residence period. We show, however, that a Neurog3TA.LO cell population, defined as Neurog3 transcriptionally active and Sox9+ and often containing nonimmunodetectable Neurog3 protein, has a relatively high mitotic index and prolonged epithelial residency. We propose that this endocrine-biased mitotic progenitor state is functionally separated from a pro-ductal pool and endows them with long-term capacity to make endocrine fate-directed progeny. A novel BAC transgenic Neurog3 reporter detected two types of mitotic behavior in Sox9+ Neurog3TA.LO progenitors, associated with progenitor pool maintenance or derivation of endocrine-committed Neurog3HI cells, respectively. Moreover, limiting Neurog3 expression dramatically increased the proportional representation of Sox9+ Neurog3TA.LO progenitors, with a doubling of its mitotic index relative to normal Neurog3 expression, suggesting that low Neurog3 expression is a defining feature of this cycling endocrine-biased state. We propose that Sox9+ Neurog3TA.LO endocrine-biased progenitors feed production of Neurog3HI endocrine-committed cells during pancreas organogenesis. [ABSTRACT FROM AUTHOR]

Neurog3-induced Dll1 expression in pancreatic endocrine progenitors ostensibly activates Hes1 expression via Notch and thereby represses Neurog3 and endocrine differentiation in neighboring cells by lateral inhibition. Here we show in mouse that Dll1 and Hes1 expression deviate during regionalization of early endoderm, and later during early pancreas morphogenesis. At that time, Ptf1a activates Dll1 in multipotent pancreatic progenitor cells (MPCs), and Hes1 expression becomes Dll1 dependent over a brief time window. Moreover, Dll1, Hes1 and Dll1/Hes1 mutant phenotypes diverge during organ regionalization, become congruent at early bud stages, and then diverge again at late bud stages. Persistent pancreatic hypoplasia in Dll1 mutants after eliminating Neurog3 expression and endocrine development, together with reduced proliferation of MPCs in both Dll1 and Hes1 mutants, reveals that the hypoplasia is caused by a growth defect rather than by progenitor depletion. Unexpectedly, we find that Hes1 is required to sustain Ptf1a expression, and in turn Dll1 expression in early MPCs. Our results show that Ptf1a-induced Dll1 expression stimulates MPC proliferation and pancreatic growth by maintaining Hes1 expression and Ptf1a protein levels. [ABSTRACT FROM AUTHOR]