Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of mouse Ngn3-expressing pancreatic cells in a time series through embryonic development and adult islet cells

ABSTRACT: Neurogenin3 (Ngn3) is a basic helix-loop-helix transcription factor which is expressed in scattered cells in the embryonic pancreas. Mice deficient for Ngn3 fail to produce any pancreatic endocrine cells and die shortly after birth. Expression of transcription factors critical to pancreatic development (Isl1, NeuroD, Pax4, and Pax6) is missing and endocrine precursors are absent in mutant pancreatic epithelium. This study utilizes mice which were engineered to contain EGFP (Enhanced Green Fluorescent Protein) under the control of the Ngn3 promoter. In this way, cells which express Ngn3 can be FACS sorted and studied throughout embryonic development (E13.5, E14.5, E15.5, E16.5, and E17.5). EGFP positive cells from the embryonic time-points in addition to adult islets (no cells expressing Ngn3) were hybridized to the BCBC PancChip 6.0 expression microarray, thus generating a profile of gene expression during this critical stage of development of the endocrine pancreas.

ORGANISM(S): Mus musculus

SUBMITTER: Peter White

PROVIDER: E-CBIL-40 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Aims/hypothesis: Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it. Methods: The extent of the Ngn3-mediated duct-to-endocrine cell reprogramming was measured employing genome wide mRNA profiling. By modulation of the Delta-Notch signaling or addition of pancreatic endocrine transcription factors Myt1, MafA and Pdx1 we intended to improve the reprogramming. Results: Ngn3 stimulates duct cells to express a focused set of genes that are abundant in islet endocrine cells and/or neural tissues. This neuro-endocrine shift, however, covers a minor fraction (5%) of the estimated genome-wide transcriptome difference between duct and islet endocrine cells. Interestingly, transduction of exogenous Ngn3 activates endogenous Ngn3 suggesting auto-activation of this gene. Furthermore, pancreatic endocrine reprogramming of human duct cells can be moderately enhanced by inhibition of Delta-Notch signaling as well as by co-expressing the transcription factor Myt1, but not MafA and Pdx1. Conclusions/interpretation: The results provide further insight into the plasticity of adult human duct cells and suggest measurable routes to enhance Ngn3-mediated in vitro reprogramming protocols for regenerative beta cell therapy in diabetes. We used Affymetrix HG133A and HG133B to get a comprehensive view on the reprogramming potential in vitro of human pancreatic duct cell cultures (n=3-4) at 3 and 14/20 days after ectopic adenoviral expression of murine neurogenin 3 as compared to GFP-expressing control vectors. The microarray analysis was performed on 3 independent samples that each contained RNA extracted from a pool of 3 independent donor pancreata. The total number of non-selected donor organs is 9. Transcripts were considered as differentially regulated by Ngn3 when 1.5 fold (LCB, unpaired P < 0.05) up- or down-regulated in AdGFP-Ngn3 versus AdGFP controls, at 3 and/or 14 dpi. Transcripts that showed differential expression between day 3 and day 14 in AdGFP-Ngn3 duct cells but not in AdGFP control cells, were also considered Ngn3-regulated

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Transcription profiling of mouse Ngn3-expressing pancreatic cells in a time series through embryonic development and adult islet cells

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