Abstract
SUMMARYTransient expression of the transcription factor neurogenin-3 marks progenitor cells in the pancreas as they differentiate into islet cells. We developed a transgenic mouse line in which the surrogate markers secreted alkaline phosphatase (SeAP) and enhanced green florescent protein (EGFP) can be used to monitor neurogenin-3 expression, and thus islet cell genesis. In transgenic embryos, cells expressing EGFP lined the pancreatic ducts. SeAP was readily detectable in embryos, in the media of cultured embryonic pancreases and in the serum of adult animals. Treatment with the γ-secretase inhibitor DAPT, which blocks Notch signaling, enhanced SeAP secretion rates and increased the number of EGFP-expressing cells as assayed by fluorescence-activated cell sorting (FACS) and immunohistochemistry in cultured pancreases from embryos at embryonic day 11.5, but not in pancreases harvested 1 day later. By contrast, treatment with growth differentiation factor 11 (GDF11) reduced SeAP secretion rates. In adult mice, partial pancreatectomy decreased, whereas duct ligation increased, circulating SeAP levels. This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.
Highlights
Therapeutic methods for generating new insulin-producing islet cells remain an unrealized goal of diabetes treatment
Generation of transgenic BAC NEUROG3-secreted alkaline phosphatase (SeAP)/enhanced green florescent protein (EGFP) mice To produce a transgene that would allow for the assessment of neurogenin-3 gene expression in vivo, we started with a BAC containing the human genomic sequence 134-kb upstream and 30kb downstream of the human neurogenin-3 (NEUROG3) gene, and replaced the coding sequence for neurogenin-3 with two marker genes, SeAP and EGFP, separated by a viral internal ribosomal entry site (IRES) (Fig. 1A)
The transgenes are expressed in the endocrine lineage during development To characterize transgene expression, we compared it to the expression of native neurogenin-3 RNA and protein
Summary
Therapeutic methods for generating new insulin-producing islet cells remain an unrealized goal of diabetes treatment. Models by which these processes can be tracked in vivo can provide the means for testing methods for manipulating islet cell generation. The exocrine, endocrine and duct cells differentiate from these undifferentiated pancreatic progenitor cells (Slack, 1995; Wilson et al, 2003). Understanding and controlling this process of differentiation could provide us with the cells needed to treat diabetes mellitus. Insulin deficiency underlies both major forms of diabetes. Therapies that enable replacement of b-cells are needed for both type 1 and type 2 diabetes, and we need animal models for identifying and testing these therapies
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