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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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The SeAP transgene is expressed in the endocrine lineage the embryonic pancreas of BAC NEUROG3-SeAP/EGFP transgenic mice. (A,B) Enzymatic SeAP activity (purple) was colocalized with immunohistochemical staining of endogenous neurogenin-3 (brown, A) in E14.5 fetal pancreases or ductal marker DBA lectin (brown, B) in E18.5 fetal pancreases. Scale bar: 50 μm. (C) Pancreases were harvested from E12.5 embryos and placed in culture at day 1. At 24-hour intervals, SeAP activity in the media was assayed [reported as relative light units (RLUs)], and media was replaced. The solid line indicates media cultured with transgenic pancreases; the dashed line indicates media cultured with non-transgenic pancreases. Data represent mean ± s.e.m. from six pancreases in each group.
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f4-0040268: The SeAP transgene is expressed in the endocrine lineage the embryonic pancreas of BAC NEUROG3-SeAP/EGFP transgenic mice. (A,B) Enzymatic SeAP activity (purple) was colocalized with immunohistochemical staining of endogenous neurogenin-3 (brown, A) in E14.5 fetal pancreases or ductal marker DBA lectin (brown, B) in E18.5 fetal pancreases. Scale bar: 50 μm. (C) Pancreases were harvested from E12.5 embryos and placed in culture at day 1. At 24-hour intervals, SeAP activity in the media was assayed [reported as relative light units (RLUs)], and media was replaced. The solid line indicates media cultured with transgenic pancreases; the dashed line indicates media cultured with non-transgenic pancreases. Data represent mean ± s.e.m. from six pancreases in each group.

Mentions: SeAP was readily detected not only by measurement of enzymatic activity in serum and pancreatic extracts from the transgenic fetuses, but also by direct histological detection of SeAP activity with chromogenic substrate NBT/BCIP in pancreatic sections at E14.5 and E18.5 (Fig. 4A,B). Interestingly, SeAP staining with NBT/BCIP colocalized with neurogenin-3 protein more closely than did EGFP (no cells lacking neurogenin-3 protein had robust SeAP activity).


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)

The SeAP transgene is expressed in the endocrine lineage the embryonic pancreas of BAC NEUROG3-SeAP/EGFP transgenic mice. (A,B) Enzymatic SeAP activity (purple) was colocalized with immunohistochemical staining of endogenous neurogenin-3 (brown, A) in E14.5 fetal pancreases or ductal marker DBA lectin (brown, B) in E18.5 fetal pancreases. Scale bar: 50 μm. (C) Pancreases were harvested from E12.5 embryos and placed in culture at day 1. At 24-hour intervals, SeAP activity in the media was assayed [reported as relative light units (RLUs)], and media was replaced. The solid line indicates media cultured with transgenic pancreases; the dashed line indicates media cultured with non-transgenic pancreases. Data represent mean ± s.e.m. from six pancreases in each group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4-0040268: The SeAP transgene is expressed in the endocrine lineage the embryonic pancreas of BAC NEUROG3-SeAP/EGFP transgenic mice. (A,B) Enzymatic SeAP activity (purple) was colocalized with immunohistochemical staining of endogenous neurogenin-3 (brown, A) in E14.5 fetal pancreases or ductal marker DBA lectin (brown, B) in E18.5 fetal pancreases. Scale bar: 50 μm. (C) Pancreases were harvested from E12.5 embryos and placed in culture at day 1. At 24-hour intervals, SeAP activity in the media was assayed [reported as relative light units (RLUs)], and media was replaced. The solid line indicates media cultured with transgenic pancreases; the dashed line indicates media cultured with non-transgenic pancreases. Data represent mean ± s.e.m. from six pancreases in each group.
Mentions: SeAP was readily detected not only by measurement of enzymatic activity in serum and pancreatic extracts from the transgenic fetuses, but also by direct histological detection of SeAP activity with chromogenic substrate NBT/BCIP in pancreatic sections at E14.5 and E18.5 (Fig. 4A,B). Interestingly, SeAP staining with NBT/BCIP colocalized with neurogenin-3 protein more closely than did EGFP (no cells lacking neurogenin-3 protein had robust SeAP activity).

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