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.


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.
Miyatsuka
Takeshi
T
Diabetes Center and Department of Medicine, University of California, San Francisco, San Francisco, California, USA.
Li
Zhongmei
Z
German
Michael S
MS
D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics* / Q000502:physiology; D002454:Cell Differentiation; D005434:Flow Cytometry / Q000379:methods; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D005934:Glucagon / Q000235:genetics; D007328:Insulin / Q000235:genetics; D007515:Islets of Langerhans / Q000166:cytology* / Q000502:physiology; D007700:Kinetics; D051379:Mice; D023421:Models, Animal; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction
D051792:Basic Helix-Loop-Helix Proteins; D005934:Glucagon; D007328:Insulin; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; C407737:Neurog3 protein, mouse