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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.

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Time-dependent shift of fluorescence in sorted cells after culture. Ngn3-Timer pancreata were dissociated at E17.5 and sorted by FACS. The sorted cells from the gates shown were analyzed immediately after FACS by flow cytometry (first column) or placed in culture and analyzed by flow cytometry after 3 h (second column), 6 h (third column), and 12 h (fourth column).
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Figure 2: Time-dependent shift of fluorescence in sorted cells after culture. Ngn3-Timer pancreata were dissociated at E17.5 and sorted by FACS. The sorted cells from the gates shown were analyzed immediately after FACS by flow cytometry (first column) or placed in culture and analyzed by flow cytometry after 3 h (second column), 6 h (third column), and 12 h (fourth column).

Mentions: To verify this hypothesis and estimate the temporal resolution of this model, fluorescent cells were sorted by a fluorescence-activated cell sorter (FACS) into four different populations, placed in culture, and then reanalyzed by flow cytometry at various time points following culture. Green-dominant cells, sorted from gate A in Fig. 2, converted to green/red double-positive within 6 h, whereas green/red double-positive cells in gate B converted to the lower green/red ratio (red dominant) of gate C cells within 12 h. Therefore, the green-dominant cells were within a 6-h time window after initial DsRed-E5 expression and green/red double-positive cells within a 12-h time window. On the other hand, the fluorescent cells sorted using gate C changed little over 12 h, presumably because of the long half-life of DsRed-E5.


Chronology of islet differentiation revealed by temporal cell labeling.

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

Time-dependent shift of fluorescence in sorted cells after culture. Ngn3-Timer pancreata were dissociated at E17.5 and sorted by FACS. The sorted cells from the gates shown were analyzed immediately after FACS by flow cytometry (first column) or placed in culture and analyzed by flow cytometry after 3 h (second column), 6 h (third column), and 12 h (fourth column).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Time-dependent shift of fluorescence in sorted cells after culture. Ngn3-Timer pancreata were dissociated at E17.5 and sorted by FACS. The sorted cells from the gates shown were analyzed immediately after FACS by flow cytometry (first column) or placed in culture and analyzed by flow cytometry after 3 h (second column), 6 h (third column), and 12 h (fourth column).
Mentions: To verify this hypothesis and estimate the temporal resolution of this model, fluorescent cells were sorted by a fluorescence-activated cell sorter (FACS) into four different populations, placed in culture, and then reanalyzed by flow cytometry at various time points following culture. Green-dominant cells, sorted from gate A in Fig. 2, converted to green/red double-positive within 6 h, whereas green/red double-positive cells in gate B converted to the lower green/red ratio (red dominant) of gate C cells within 12 h. Therefore, the green-dominant cells were within a 6-h time window after initial DsRed-E5 expression and green/red double-positive cells within a 12-h time window. On the other hand, the fluorescent cells sorted using gate C changed little over 12 h, presumably because of the long half-life of DsRed-E5.

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