Pancreas-specific primary regulatory targets of Nkx2.2

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DESCRIPTION (provided by applicant): Homeobox genes of the Nkx2.2/vnd family are required for pancreatic specification and ventral neural tube patterning. This gene family plays conserved roles in lineage specification in divergent animal species, including flies and man. Although invertebrates lack a pancreas, endocrine cells in the nervous and digestive systems secrete insulin to co-ordinate cellular metabolism and nutritional conditions. Seven insulin-like genes have recently been described in Drosophila. The pancreatic and neural lineages are ancestrally related, and share common regulatory factors. In mice, the insulin enhancer drives expression in the ventral neural tube and the pancreas, while fly neurons produce insulin. Moreover, ES cells, exposed to factors that generate neurons, develop into both motor neurons and islets. Nkx2.2/Vnd-type transcription factors are critical for aspects of pancreatic and neural specification, yet how this family functions is not understood, primarily because target genes remain largely unidentified. We previously described, ind, a dorsal-ventral patterning gene that is directly repressed by Drosophila Vnd. This is to date the only well-characterized target of the Nkx2.2/Vnd family. The simplicity, yet sophistication of the fruit fly, Drosophila, as a genetic model has provided novel insights into aspects of development ranging from insulin responsiveness to aging. Here we propose identifying targets of the Nkx2.2/vnd gene family using microarray analyses on mRNAs isolated from Drosophila embryos that transiently over-express vnd at times when only primary (direct) target genes should be activated. To assess the feasibility of this pilot project, initially we will focus upon those candidate vnd target genes that have previously been studied by other labs. Quantitative RT-PCR and in situ hybridization on RNAs from wild-type, vnd minus, and vnd over-expression mutant embryos will further establish whether the candidate vnd target genes are real targets. To address whether vertebrate homologues of the target fly genes we identify are critical for pancreatic development, expression of conserved vertebrate homologues will be assessed in the developing pancreas of wild-type and NKx2.2 mutants. This pilot study is designed to exploit the versatile Drosophila model to gain pioneering insights into NKx2.2 target genes. This knowledge may be highly instructive in defining those regulatory networks required for beta islet specification.