The basic helix-loop-helix transcription factor Neurogenin3 (Ngn3/Neurog3) is expressed in endocrine progenitor cells in the embryonic mouse pancreas. Ngn3 controls endocrine cell fate decisions. Ngn3 deficient mice do not develop any pancreatic endocrine cells, including insulin producing beta cells, and die postnatally from diabetes. Therefore, the characterization of gene expression in Ngn3-expressing cells and their progeny is of particular interest for the development of novel strategies for cell replacement therapies in type-1 diabetes. Here we describe two studies. In the first study (8 assays) we used mice where the EYFP (Enhanced Yellow fluorescent Protein) is expressed under the control of Ngn3 regulatory elements (knock add on strategy). EYFP-positive, Ngn3-expressing cells, were FACS sorted from embryonic pancreas at day 15.5 (E15.5), as well as EYFP-negative cells. In the second study (6 assays) we compared wild-type and Ngn3 mutant pancreas at E15.5. All samples were hybridized to Affymetrix GeneChip Mouse Genome 430.2.0 array.

Determine the gene expression profile of islet progenitor cells and identify potential novel downstream effectors of Neurog3 (neurogenin 3) function.

Single cell suspensions of eYFP-positive and negative cells were prepared from E15.5 Ngn3 eYFP/+ mice. Microarray analysis of biological quadruplicates was performed using Affymetrix GeneChip Mouse Genome 430 2.0 arrays. The role of zebrafish rfx6 (si:dkeyp-93d12.2) in endocrine development was examined using morpholinos.

Gene expression profiling in isolated Neurog3-positive progenitor cells resulted in the identification of 47 up-regulated transcription factors in eYFP-positive versus negative cells. These included known (Neurod1, Pax4, Pax6, Insm1 and Mafa) and previously unreported (Fev, Mlxipl, Vdr, Dach1, Lhx1, and Rfx6) transcription factors in islet development. Rfx6 was highly enriched and further characterized by in situ hybridization and immunohistochemistry experiments which showed that Rfx6 expression in the embryonic pancreas from E10.5 is restricted to developing islet cells. Morpholino injections targeting rfx6 (si:dkeyp-93d12.2) in zebrafish led to complete depletion or drastic reduction in glucagon, ghrelin, and somatostatin-expressing cells but the number of insulin-expressing cells was only slightly decreased.

Rfx6 is a novel regulator of islet cell development and is required for glucagon, ghrelin, and somatostatin-expressing cells. Absence of Rfx6 only slightly reduces beta cell numbers.

• Ngn3: 1110, 1470, 2520, 2800, 3021, 3100, 3425, 3733, 3795, 4195, 4374,
• pancreatic development: 3061, 3100, 3733, 3935, 4014, 4195, 4275, 4294, 4355, 4374, 4534,