Neurogenin3 (Ngn3) is a basic helix-loop-helix transcription factor which is expressed in scattered cells in the embryonic pancreas. Mice deficient for Ngn3 fail to produce any pancreatic endocrine cells and die shortly after birth. Expression of transcription factors critical to pancreatic development (Isl1, NeuroD, Pax4, and Pax6) is missing and endocrine precursors are absent in mutant pancreatic epithelium. This study utilizes mice which were engineered to contain EGFP (Enhanced Green Fluorescent Protein) under the control of the Ngn3 promoter. In this way, cells which express Ngn3 can be FACS sorted and studied throughout embryonic development (E13.5, E14.5, E15.5, E16.5, and E17.5). EGFP positive cells from the embryonic time-points in addition to adult islets (no cells expressing Ngn3) were hybridized to the BCBC PancChip 6.0 expression microarray, thus generating a profile of gene expression during this critical stage of development of the endocrine pancreas.

Identify potential novel targets of Ngn3 and generate a comprehensive gene expression profile of endocrine progenitor cells and their immediate descendants from embryonic pancreata.

A Neurog3-EGFP knock-in mouse model was used to isolate endocrine progenitor cells from embryonic pancreata (E13.5 through E17.5) capitalizing on the dependence of differentiation toward the endocrine lineage on the transcription factor Ngn3. Neurog3-EGFP endocrine precursors and their immediate descendants from fetal pancreas were isolated by flow cytometry taking advantage of the fact that EGFP protein persists in cells for several days after the promoter driving its expression has been turned off. RNA were analyzed on Mouse PancChip 6.0. Gene expression profiles were confirmed using qRT-PCR.

A total of 1,029 genes were identified as being temporally regulated in the endocrine lineage during fetal development. The most significantly temporally regulated gene was Glypican 3 (Gpc3), with a marked decrease in expression over time. 246 transcription factors, transcriptional regulators and potential transcriptional regulators were found to be temporally controlled during development of the endocrine precursors, several of which are well-established and important regulators of the endocrine pancreas. Regulatory networks were modeled involving these proteins which highlight the complex transcriptional hierarchy governing endocrine differentiation.

The list of temporally regulated genes identified in fetal endocrine precursors and their immediate descendants provides a novel and important resource for developmental biologists and diabetes researchers alike.

• Ngn3: 1110, 1470, 2520, 2800, 3021, 3100, 3425, 3733, 3795, 4195, 4374,