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A mouse model for monitoring islet cell genesis and developing therapies for diabetes.

Shimajiri Y, Kosaka Y, Scheel DW, Lynn FC, Kishimoto N, Wang J, Zhao S, German MS - Dis Model Mech (2010)

Bottom Line: 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.This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, University of California San Francisco, San Francisco, CA 94143-0534, USA.

ABSTRACT
Transient 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.

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GDF11 and Notch signaling regulate the SeAP transgene in fetal pancreas explants. Regulation of SeAP secretion from cultured fetal pancreases from NEUROG3-SeAP/EGFP transgenic mice. (A) Pancreases were harvested from E12.5 embryos and treated with 250 ng/ml GDF11 (dashed lines) or control media (solid lines) for 6 days. SeAP activity was assayed on the days of culture shown. Data represent mean ± s.e.m. from three pancreases in each group. (B,C) Pancreases were harvested from E11.5 embryos (B) or E12.5 embryos (C), placed in culture and treated with the γ-secretase inhibitor DAPT at 50 mM in 0.1% DMSO (black bars) or 0.1% DMSO alone (white bars). SeAP activity was assayed after 48 hours of treatment. Data represent mean + s.e.m. for the number of cultured pancreases shown.
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f6-0040268: GDF11 and Notch signaling regulate the SeAP transgene in fetal pancreas explants. Regulation of SeAP secretion from cultured fetal pancreases from NEUROG3-SeAP/EGFP transgenic mice. (A) Pancreases were harvested from E12.5 embryos and treated with 250 ng/ml GDF11 (dashed lines) or control media (solid lines) for 6 days. SeAP activity was assayed on the days of culture shown. Data represent mean ± s.e.m. from three pancreases in each group. (B,C) Pancreases were harvested from E11.5 embryos (B) or E12.5 embryos (C), placed in culture and treated with the γ-secretase inhibitor DAPT at 50 mM in 0.1% DMSO (black bars) or 0.1% DMSO alone (white bars). SeAP activity was assayed after 48 hours of treatment. Data represent mean + s.e.m. for the number of cultured pancreases shown.

Mentions: To test whether potential regulators of neurogenin-3 could influence expression of the transgene in vitro, we first measured SeAP activity in the media of fetal pancreatic bud harvested at E12.5 and cultured with TGFβ family member GDF11. Treatment with GDF11 for 4–6 days strongly inhibited SeAP secretion (Fig. 6A).


A mouse model for monitoring islet cell genesis and developing therapies for diabetes.

Shimajiri Y, Kosaka Y, Scheel DW, Lynn FC, Kishimoto N, Wang J, Zhao S, German MS - Dis Model Mech (2010)

GDF11 and Notch signaling regulate the SeAP transgene in fetal pancreas explants. Regulation of SeAP secretion from cultured fetal pancreases from NEUROG3-SeAP/EGFP transgenic mice. (A) Pancreases were harvested from E12.5 embryos and treated with 250 ng/ml GDF11 (dashed lines) or control media (solid lines) for 6 days. SeAP activity was assayed on the days of culture shown. Data represent mean ± s.e.m. from three pancreases in each group. (B,C) Pancreases were harvested from E11.5 embryos (B) or E12.5 embryos (C), placed in culture and treated with the γ-secretase inhibitor DAPT at 50 mM in 0.1% DMSO (black bars) or 0.1% DMSO alone (white bars). SeAP activity was assayed after 48 hours of treatment. Data represent mean + s.e.m. for the number of cultured pancreases shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3046103&req=5

f6-0040268: GDF11 and Notch signaling regulate the SeAP transgene in fetal pancreas explants. Regulation of SeAP secretion from cultured fetal pancreases from NEUROG3-SeAP/EGFP transgenic mice. (A) Pancreases were harvested from E12.5 embryos and treated with 250 ng/ml GDF11 (dashed lines) or control media (solid lines) for 6 days. SeAP activity was assayed on the days of culture shown. Data represent mean ± s.e.m. from three pancreases in each group. (B,C) Pancreases were harvested from E11.5 embryos (B) or E12.5 embryos (C), placed in culture and treated with the γ-secretase inhibitor DAPT at 50 mM in 0.1% DMSO (black bars) or 0.1% DMSO alone (white bars). SeAP activity was assayed after 48 hours of treatment. Data represent mean + s.e.m. for the number of cultured pancreases shown.
Mentions: To test whether potential regulators of neurogenin-3 could influence expression of the transgene in vitro, we first measured SeAP activity in the media of fetal pancreatic bud harvested at E12.5 and cultured with TGFβ family member GDF11. Treatment with GDF11 for 4–6 days strongly inhibited SeAP secretion (Fig. 6A).

Bottom Line: 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.This model will be useful for studying signals involved in islet cell genesis in vivo and developing therapies that induce this process.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center, University of California San Francisco, San Francisco, CA 94143-0534, USA.

ABSTRACT
Transient 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.

Show MeSH