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Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq of Human Embryonic Stem Cell derived pancreas progenitor differentiation, Day 13 of wildtype, HES1-/-, NEUROG3-/- and HES1-/-NEUROG3-/- genotypes

ABSTRACT: To better understand the mechanism by which HES1 regulates pancreas development we took advantage of recent developments in directed differentiation of human embryonic stem cells (hESCs) to the pancreatic endocrine lineage via a series of progenitor stages (Figure 1 and (Rezania et al., 2014). Using the iCRISPR platform (González et al., 2014), we introduced indels in the HES1 and NEUROG3 genes, either singly or in combination in iCRISPR H1 cells. Using previously described gRNAs (Zhu et al., 2016) to target exon2 of the NEUROG3 gene and exon2 of the HES1 gene we detected by Sanger and RNA-sequencing a 2 bp insertion and a T insertion, respectively, resulting in premature STOP codons in two/multiple clonal cell lines carrying the introduced mutation and the loss of NEUROG3 by immunostaining We then subjected two or more clonal lines of H1-iCRISPR (wildtype), HES1−/−, NEUROG3−/− and HES1−/−NEUROG3−/− (abbreviated H1N3-dKO or H1−/−N3−/−) to differentiation to β-like cells using a modified version of the protocol from Rezania et al. (2014). Marker analysis showed that all cell lines maintained pluripotency (OCT4+) as hESC and were able to differentiate to FOXA2+ and SOX17+ co-positive definitive endoderm (DE, Day 3), PDX1+ cells at the posterior foregut stage (PF, Day 7), to bipotent progenitors marked by PDX1+ and NKX6-1+ at the Pancreatic Endoderm stage (PE, Day 10), and the Endocrine Precursor stage (EP, Day 13)

INSTRUMENT(S): Qiacube, NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Palle Serup

PROVIDER: E-MTAB-7185 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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RNA-seq of Pdx1-GFP+ sorted E10.5, wildtype and Hes1-/-. Neurog3-dependent Pancreas Dysgenesis Causes Ectopic Pancreas in Hes1 Mutants

Project description:RNA-seq of FACS Sorted E10.5 Pdx1-GFP+ of genotypes wildtype and Hes1-/-. Summary statement The developmental mechanisms that cause ectopic pancreas are poorly understood. We show that aberrant dorsal pancreas morphogenesis in Hes1 mutants leads to ectopic pancreas depending on the pro-endocrine gene Neurog3. Abstract 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 in mouse embryos to reveal an endodermal requirement for Hes1 and that most ectopic pancreas tissue is derived from the E8.5 dorsal pancreas primordium. RNA-seq data from sorted E10.5 Pdx1-GFP+ cells from Hes1+/+ and Hes1−/− suggested that upregulation of endocrine lineage genes in Hes1−/− embryos was the major defect in the endoderm and accordingly early pancreas morphogenesis was normalised and the ectopic pancreas phenotype suppressed in Hes1−/−Neurog3−/− embryos. Analysis of other Notch pathway mutants uncovered a total depletion of progenitors in Mib1 deficient dorsal anlage, 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.

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RNA-seq of Human Embryonic Stem Cell derived pancreas progenitor differentiation, Day 13 of wildtype, HES1-/-, NEUROG3-/- and HES1-/-NEUROG3-/- genotypes

Project description:RNA-seq of Human Embryonic Stem Cell derived pancreas progenitor differentiation, Day 13 of wildtype, HES1-/-, NEUROG3-/- and HES1-/-NEUROG3-/- genotypes

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