This experiment was designed to analyze the expression of genes in dorsal pancreatic cells at two temporally separated stages of pancreas development. This was accomplished by comparing expression profiles of embryonic dorsal pancreas tissue from Ngn3 null mice with wild-type littermates at days 13 and 15 of embryonic development. The comparison of gene expression in mutant and wild-type pancreas was used primarily to show genes that are lower expressed/missing in the mutant, as Ngn3 null mice have no endocrine pancreas tissue. From each developmental stage, five wild-type and five mutant samples were chosen, representing embryos from at least three different litters. Wild-type and mutant samples from the common stage of development were paired randomly and analysed in flipped colour. Probes were spotted in duplicate on each slide in a randomised (fixed) layout, effectively distributing the duplicate spots randomly over the slide.

The aim of this study is to identify genes expressed in endocrine cells and their precursors (beginning and end of the secondary transition) using an Ngn3 knockout mouse and to map important pathways that operate during the formation of the endocrine cells.

Pancreatic gene expression was compared between Ngn3-deficient mice and littermate controls. The ArrayTAG 20k murine gene collection containing 20,188 spotted cDNAs covering 12,140 genes plus ESTs was used to compare a major part of the transcriptome in dorsal pancreas buds from wild-type and Ngn3-deficient embryos isolated at e13 and e15. Note that several pancreas-related genes including insulin, glucagon, Ngn3, Pdx1, NeuroD, Isl1 and Pax4 are not present on the array. From each developmental stage, five wild-type and five mutant samples were chosen, representing embryos from at least three different litters. Wild-type and mutant samples from the common stage of development were paired randomly and analysed in flipped colour.

Microarray analysis identified 52 genes with significant differences in expression and at least twofold reduction in Ngn3 knockouts compared to controls. Many of them were previously described to be involved in endocrine development and function. Among the genes not previously characterized were Rhomboid veinlet-like 4, genes involved in tetrahydrobiopterin biosynthesis and the Iroquois-type homeobox gene Irx1, the latter was selected for further investigation. In situ hybridisation demonstrated that two Iroquois genes, Irx1 and Irx2, were expressed in pancreatic endoderm of wild-type, but not Ngn3 mutant embryos. Furthermore, ectopic Ngn3 induced prominent Irx2 expression in chicken endoderm. Co-labelling established that Irx1 and Irx2 mRNA is located to glucagon-, but not insulin- or somatostatin-producing cells in mice and chicken.

These data suggest that Irx1 and Irx2 serve an evolutionary conserved role in the regulation of a-cell-specific gene expression.

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