Chronology of islet differentiation revealed by temporal cell labeling.
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.
Pubmed ID: 19478145
August 29, 2009
- Agency: NIDDK NIH HHS, Id: P30 DK63720
- Agency: NIDDK NIH HHS, Id: R01 DK021344
- Basic Helix-Loop-Helix Transcription Factors
- Cell Differentiation
- Flow Cytometry
- Gene Expression Profiling
- Genes, Reporter
- Islets of Langerhans
- Models, Animal
- Nerve Tissue Proteins
- RNA, Messenger
- Reverse Transcriptase Polymerase Chain Reaction