Limits...
Chronology of islet differentiation revealed by temporal cell labeling.

Miyatsuka T, Li Z, German MS - Diabetes (2009)

Bottom Line: When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h.The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas.The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, California, USA.

ABSTRACT

Objective: Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene.

Research design and methods: In order to separate the transient neurogenin 3 -expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus.

Results: In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas.

Conclusions: The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells.

Show MeSH
Expression of the DsRed-E5 fluorescent protein in developing pancreas. A: The pancreas was dissected from an Ngn3-Timer embryo at E17.5 and stained with DBA lectin, a marker of pancreatic duct (blue). Green-dominant cells detected within ducts are labeled with white arrows; green/red double-positive and red-dominant cells (yellow or orange in color, right panel) are labeled with orange arrowheads. Scale bar: 50 μm. B: Pancreata from wild-type (A–C) and Ngn3-Timer mice (d–F) were dissociated at E13.5, E17.5, and P0 and analyzed using flow cytometry. Green fluorescence is shown on the vertical axis and red fluorescence on the horizontal axis. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2712795&req=5

Figure 1: Expression of the DsRed-E5 fluorescent protein in developing pancreas. A: The pancreas was dissected from an Ngn3-Timer embryo at E17.5 and stained with DBA lectin, a marker of pancreatic duct (blue). Green-dominant cells detected within ducts are labeled with white arrows; green/red double-positive and red-dominant cells (yellow or orange in color, right panel) are labeled with orange arrowheads. Scale bar: 50 μm. B: Pancreata from wild-type (A–C) and Ngn3-Timer mice (d–F) were dissociated at E13.5, E17.5, and P0 and analyzed using flow cytometry. Green fluorescence is shown on the vertical axis and red fluorescence on the horizontal axis. (A high-quality digital representation of this figure is available in the online issue.)

Mentions: To solve this problem, we developed a novel transgenic mouse model (Ngn3-Timer) in which human NEUROG3 upstream and downstream sequences were used within a bacterial artificial chromosome (BAC) to drive expression of DsRed-E5, a variant of the Discosoma sp. red fluorescent protein that shifts its fluorescence emission peak from green to red in a time-dependent manner (16). Using fluorescence microscopy, green fluorescence could be detected in developing pancreata of Ngn3-Timer embryos as early as embryonic day 9.5 (E9.5) (data not shown). Both green and red fluorescent signals were readily detected in developing pancreata of Ngn3-Timer embryos from E12.5 to E18.5, whereas predominantly red fluorescence was observed at postnatal day 7 (P7) (supplemental Fig. 1, available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0390/DC1), consistent with previous reports that few Neurog3-expressing cells persist after birth (5). At E17.5, histological analyses detected green-dominant and green/red double-positive fluorescent cells in close apposition with the ductal lumen, whereas red-dominant cells appeared in islet-like clusters (Fig. 1A), supporting a model whereby cells in the endocrine lineage emerge from ductal regions as Neurog3-positive cells and migrate away from the ductal region as differentiation progresses. Consistent with this model, staining for Neurog3 protein overlapped with predominantly green-dominant cells and some green/red double-positive cells (supplemental Fig. 2), whereas insulin staining overlapped with green/red double-positive cells and red-dominant cells (supplemental Fig. 3).


Chronology of islet differentiation revealed by temporal cell labeling.

Miyatsuka T, Li Z, German MS - Diabetes (2009)

Expression of the DsRed-E5 fluorescent protein in developing pancreas. A: The pancreas was dissected from an Ngn3-Timer embryo at E17.5 and stained with DBA lectin, a marker of pancreatic duct (blue). Green-dominant cells detected within ducts are labeled with white arrows; green/red double-positive and red-dominant cells (yellow or orange in color, right panel) are labeled with orange arrowheads. Scale bar: 50 μm. B: Pancreata from wild-type (A–C) and Ngn3-Timer mice (d–F) were dissociated at E13.5, E17.5, and P0 and analyzed using flow cytometry. Green fluorescence is shown on the vertical axis and red fluorescence on the horizontal axis. (A high-quality digital representation of this figure is available in the online issue.)
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2712795&req=5

Figure 1: Expression of the DsRed-E5 fluorescent protein in developing pancreas. A: The pancreas was dissected from an Ngn3-Timer embryo at E17.5 and stained with DBA lectin, a marker of pancreatic duct (blue). Green-dominant cells detected within ducts are labeled with white arrows; green/red double-positive and red-dominant cells (yellow or orange in color, right panel) are labeled with orange arrowheads. Scale bar: 50 μm. B: Pancreata from wild-type (A–C) and Ngn3-Timer mice (d–F) were dissociated at E13.5, E17.5, and P0 and analyzed using flow cytometry. Green fluorescence is shown on the vertical axis and red fluorescence on the horizontal axis. (A high-quality digital representation of this figure is available in the online issue.)
Mentions: To solve this problem, we developed a novel transgenic mouse model (Ngn3-Timer) in which human NEUROG3 upstream and downstream sequences were used within a bacterial artificial chromosome (BAC) to drive expression of DsRed-E5, a variant of the Discosoma sp. red fluorescent protein that shifts its fluorescence emission peak from green to red in a time-dependent manner (16). Using fluorescence microscopy, green fluorescence could be detected in developing pancreata of Ngn3-Timer embryos as early as embryonic day 9.5 (E9.5) (data not shown). Both green and red fluorescent signals were readily detected in developing pancreata of Ngn3-Timer embryos from E12.5 to E18.5, whereas predominantly red fluorescence was observed at postnatal day 7 (P7) (supplemental Fig. 1, available in the online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0390/DC1), consistent with previous reports that few Neurog3-expressing cells persist after birth (5). At E17.5, histological analyses detected green-dominant and green/red double-positive fluorescent cells in close apposition with the ductal lumen, whereas red-dominant cells appeared in islet-like clusters (Fig. 1A), supporting a model whereby cells in the endocrine lineage emerge from ductal regions as Neurog3-positive cells and migrate away from the ductal region as differentiation progresses. Consistent with this model, staining for Neurog3 protein overlapped with predominantly green-dominant cells and some green/red double-positive cells (supplemental Fig. 2), whereas insulin staining overlapped with green/red double-positive cells and red-dominant cells (supplemental Fig. 3).

Bottom Line: When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h.The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas.The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, California, USA.

ABSTRACT

Objective: Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. Whereas mouse models expressing reporters such as eGFP or LacZ under the control of the Neurog3 gene enable us to label cells in the pancreatic endocrine lineage, the long half-life of most reporter proteins makes it difficult to distinguish cells actively expressing neurogenin 3 from differentiated cells that have stopped transcribing the gene.

Research design and methods: In order to separate the transient neurogenin 3 -expressing endocrine progenitor cells from the differentiating endocrine cells, we developed a mouse model (Ngn3-Timer) in which DsRed-E5, a fluorescent protein that shifts its emission spectrum from green to red over time, was expressed transgenically from the NEUROG3 locus.

Results: In the Ngn3-Timer embryos, green-dominant cells could be readily detected by microscopy or flow cytometry and distinguished from green/red double-positive cells. When fluorescent cells were sorted into three different populations by a fluorescence-activated cell sorter, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double-positive cells within 6 h. The sorted cell populations were then used to determine the temporal patterns of expression for 145 transcriptional regulators in the developing pancreas.

Conclusions: The precise temporal resolution of this model defines the narrow window of neurogenin 3 expression in islet progenitor cells and permits sequential analyses of sorted cells as well as the testing of gene regulatory models for the differentiation of pancreatic islet cells.

Show MeSH