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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.

Show MeSH
Temporal transcriptome analysis in the pancreata of Ngn3-Timer embryos. A–D: Ngn3-Timer pancreata were dissected at E17.5 and sorted by FACS into four gates (gates A, B, C, and N). The sorted cell populations were analyzed by real-time RT-PCR for mRNA-encoding transcriptional regulators and endocrine hormones. All expression levels were normalized to β-glucuronidase. Neurog3 (A), insulin1 (B), insulin2 (C), glucagon (D). E–M: TaqMan array was performed for 145 transcription factors. Expression levels are shown relative to the level in gate N. Each data point represents the mean of three independent experiments.
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Figure 3: Temporal transcriptome analysis in the pancreata of Ngn3-Timer embryos. A–D: Ngn3-Timer pancreata were dissected at E17.5 and sorted by FACS into four gates (gates A, B, C, and N). The sorted cell populations were analyzed by real-time RT-PCR for mRNA-encoding transcriptional regulators and endocrine hormones. All expression levels were normalized to β-glucuronidase. Neurog3 (A), insulin1 (B), insulin2 (C), glucagon (D). E–M: TaqMan array was performed for 145 transcription factors. Expression levels are shown relative to the level in gate N. Each data point represents the mean of three independent experiments.

Mentions: Real-time PCR analyses performed on RNA from cells sorted by FACS into the four populations defined in Fig. 2 confirmed the sequential maturation of these cells. The expression level of Neurog3 was highest in green-dominant cells and dramatically decreased in green/red double-positive cells (Fig. 3A), reflecting the rapid downregulation of Neurog3 once it has initiated endocrine differentiation. Expression of the islet hormones peaked later (Fig. 3B–D), with the mRNA encoding insulin and glucagon highest in cells from gate C, the most mature population.


Chronology of islet differentiation revealed by temporal cell labeling.

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

Temporal transcriptome analysis in the pancreata of Ngn3-Timer embryos. A–D: Ngn3-Timer pancreata were dissected at E17.5 and sorted by FACS into four gates (gates A, B, C, and N). The sorted cell populations were analyzed by real-time RT-PCR for mRNA-encoding transcriptional regulators and endocrine hormones. All expression levels were normalized to β-glucuronidase. Neurog3 (A), insulin1 (B), insulin2 (C), glucagon (D). E–M: TaqMan array was performed for 145 transcription factors. Expression levels are shown relative to the level in gate N. Each data point represents the mean of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Temporal transcriptome analysis in the pancreata of Ngn3-Timer embryos. A–D: Ngn3-Timer pancreata were dissected at E17.5 and sorted by FACS into four gates (gates A, B, C, and N). The sorted cell populations were analyzed by real-time RT-PCR for mRNA-encoding transcriptional regulators and endocrine hormones. All expression levels were normalized to β-glucuronidase. Neurog3 (A), insulin1 (B), insulin2 (C), glucagon (D). E–M: TaqMan array was performed for 145 transcription factors. Expression levels are shown relative to the level in gate N. Each data point represents the mean of three independent experiments.
Mentions: Real-time PCR analyses performed on RNA from cells sorted by FACS into the four populations defined in Fig. 2 confirmed the sequential maturation of these cells. The expression level of Neurog3 was highest in green-dominant cells and dramatically decreased in green/red double-positive cells (Fig. 3A), reflecting the rapid downregulation of Neurog3 once it has initiated endocrine differentiation. Expression of the islet hormones peaked later (Fig. 3B–D), with the mRNA encoding insulin and glucagon highest in cells from gate C, the most mature population.

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