Limits...
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.

Show MeSH
The EGFP transgene is expressed in the endocrine lineage in the embryonic pancreas of BAC NEUROG3-SeAP/EGFP transgenic mice. (A,B) Native EGFP fluorescence from whole-mounted pancreas at E15.5 (A) and E16.5 (B). (C) Immunofluorescent staining of whole-mounted E15.5 pancreas for mucin-1 (blue) together with immunofluorescent staining for EGFP (green). (D–I) Immunofluorescent staining of frozen E15.5 pancreatic sections for EGFP (green) and neurogenin-3 (red) together with glucagon (blue; D and E) or insulin (blue; G and H). White arrowheads mark representative EGFP positive cells co-stained with neurogenin-3. Yellow arrowheads mark representative EGFP-positive cells co-stained with glucagon or insulin. Scale bars: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3046103&req=5

f2-0040268: The EGFP transgene is expressed in the endocrine lineage in the embryonic pancreas of BAC NEUROG3-SeAP/EGFP transgenic mice. (A,B) Native EGFP fluorescence from whole-mounted pancreas at E15.5 (A) and E16.5 (B). (C) Immunofluorescent staining of whole-mounted E15.5 pancreas for mucin-1 (blue) together with immunofluorescent staining for EGFP (green). (D–I) Immunofluorescent staining of frozen E15.5 pancreatic sections for EGFP (green) and neurogenin-3 (red) together with glucagon (blue; D and E) or insulin (blue; G and H). White arrowheads mark representative EGFP positive cells co-stained with neurogenin-3. Yellow arrowheads mark representative EGFP-positive cells co-stained with glucagon or insulin. Scale bars: 50 μm.

Mentions: As expected, EGFP protein in the pancreases of BAC NEUROG3-SeAP/EGFP embryos was detected in cells along the ducts and peaked at E14.5-E15.5, shortly after the peak in neurogenin-3 mRNA (Fig. 2A and data not shown). Quantification by fluorescence-activated cell sorting (FACS) yielded 62,000 EGFP-expressing cells from 20 pancreases at E15.5, which is approximately 3000 cells per pancreas or ∼5% of the total cell population (data not shown). Although EGFP-positive cells were still present 1 day later, at E16.5, the density of EGFP-expressing cells had decreased (Fig. 2B).


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 EGFP transgene is expressed in the endocrine lineage in the embryonic pancreas of BAC NEUROG3-SeAP/EGFP transgenic mice. (A,B) Native EGFP fluorescence from whole-mounted pancreas at E15.5 (A) and E16.5 (B). (C) Immunofluorescent staining of whole-mounted E15.5 pancreas for mucin-1 (blue) together with immunofluorescent staining for EGFP (green). (D–I) Immunofluorescent staining of frozen E15.5 pancreatic sections for EGFP (green) and neurogenin-3 (red) together with glucagon (blue; D and E) or insulin (blue; G and H). White arrowheads mark representative EGFP positive cells co-stained with neurogenin-3. Yellow arrowheads mark representative EGFP-positive cells co-stained with glucagon or insulin. Scale bars: 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-0040268: The EGFP transgene is expressed in the endocrine lineage in the embryonic pancreas of BAC NEUROG3-SeAP/EGFP transgenic mice. (A,B) Native EGFP fluorescence from whole-mounted pancreas at E15.5 (A) and E16.5 (B). (C) Immunofluorescent staining of whole-mounted E15.5 pancreas for mucin-1 (blue) together with immunofluorescent staining for EGFP (green). (D–I) Immunofluorescent staining of frozen E15.5 pancreatic sections for EGFP (green) and neurogenin-3 (red) together with glucagon (blue; D and E) or insulin (blue; G and H). White arrowheads mark representative EGFP positive cells co-stained with neurogenin-3. Yellow arrowheads mark representative EGFP-positive cells co-stained with glucagon or insulin. Scale bars: 50 μm.
Mentions: As expected, EGFP protein in the pancreases of BAC NEUROG3-SeAP/EGFP embryos was detected in cells along the ducts and peaked at E14.5-E15.5, shortly after the peak in neurogenin-3 mRNA (Fig. 2A and data not shown). Quantification by fluorescence-activated cell sorting (FACS) yielded 62,000 EGFP-expressing cells from 20 pancreases at E15.5, which is approximately 3000 cells per pancreas or ∼5% of the total cell population (data not shown). Although EGFP-positive cells were still present 1 day later, at E16.5, the density of EGFP-expressing cells had decreased (Fig. 2B).

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