Abstract

Objective: Neurogenin 3 plays a pivotal role in pancreatic endocrine differentiation. While 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 make it difficult to distinguish cells actively expressing Neurogenin3 from differentiated cells that have stopped transcribing the gene.

Research Design and Methods: In order to separate the transient, Neurogenin3-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 FACS, placed in culture, and then reanalyzed by flow cytometry, green-dominant cells converted to green/red double positive within six hours. The sorted cell populations were then used to determine the temporal patterns of expression of 145 transcriptional regulators in the developing pancreas.

Conclusion: The precise temporal resolution of this model defines the narrow window of Neurogenin3 expression in islet progenitor cells and permits sequential analyses of sorted cells and the testing of gene regulatory models for the differentiation of pancreatic islet cells.