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Despite clear physiological roles, the ventromedial hypothalamus (VMH) developmental programs are poorly understood. Here, we asked whether the proneural gene achaete-scute homolog 1 ( Ascl1 ) contributes to VMH development. Ascl1 transcripts were detected in embryonic day (E) 10.5 to postnatal day 0 VMH neural progenitors. The elimination of Ascl1 reduced the number of VMH neurons at E12.5 and E15.5, particularly within the VMH-central (VMH C ) and -dorsomedial (VMH DM ) subdomains, and resulted in a VMH cell fate change from glutamatergic to GABAergic. We observed a loss of Neurog3 expression in Ascl1 -/- hypothalamic progenitors and an upregulation of Neurog3 when Ascl1 was overexpressed. We also demonstrated a glutamatergic to GABAergic fate switch in Neurog3 -null mutant mice, suggesting that Ascl1 might act via Neurog3 to drive VMH cell fate decisions. We also showed a concomitant increase in expression of the central GABAergic fate determinant Dlx1/2 in the Ascl1- null hypothalamus. However, Ascl1 was not sufficient to induce an ectopic VMH fate when overexpressed outside the normal window of competency. Combined, Ascl1 is required but not sufficient to specify the neurotransmitter identity of VMH neurons, acting in a transcriptional cascade with Neurog3 ., Competing Interests: Competing interestsD.M.K. is co-founder of Path Therapeutics, a start-up focused on anti-seizure drug discovery. The other authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

During pancreatic development, endocrine cells appear from the pancreatic epithelium when Neurog3-positive cells delaminate and differentiate into α-, β-, γ- and δ-cells. The mechanisms involved in this process are still incompletely understood. We characterized the temporal, lineage-specific developmental programs during pancreatic development by sequencing the transcriptome of thousands of individual pancreatic cells from E12.5 to E18.5 in mice, and identified all known cell types that are present in the embryonic pancreas, but focused specifically on α- and β-cell differentiation by enrichment of a MIP-GFP reporter. We characterized transcriptomic heterogeneity in the tip domain based on proliferation, and characterized two endocrine precursor clusters marked by expression of Neurog3 and Fev Pseudotime analysis revealed specific branches for developing α- and β-cells, which allowed identification of specific gene regulation patterns. These include some known and many previously unreported genes that appear to define pancreatic cell fate transitions. This resource allows dynamic profiling of embryonic pancreas development at single cell resolution and reveals novel gene signatures during pancreatic differentiation into α- and β-cells., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

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.)

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.)