Pancreas-specific primary regulatory targets of Nkx2.2

描述（由申请人提供）：胰腺规格和腹侧神经管模式需要Nkx2.2/vnd家族的同源盒基因。该基因家族在包括苍蝇和人类在内的不同动物物种的谱系规范中起保守作用。虽然无脊椎动物没有胰腺，但神经和消化系统中的内分泌细胞分泌胰岛素来协调细胞代谢和营养状况。最近在果蝇中发现了7个胰岛素样基因。胰腺和神经谱系是祖先相关的，并共享共同的调节因子。在小鼠体内，胰岛素增强剂驱动腹侧神经管和胰腺的表达，而果蝇的神经元则产生胰岛素。此外，胚胎干细胞暴露在产生神经元的因子中，既发育为运动神经元，也发育为胰岛细胞。Nkx2.2/ vnd型转录因子在胰腺和神经功能方面至关重要，但该家族的功能尚不清楚，主要是因为靶基因在很大程度上尚未确定。我们之前描述过，例如，一个直接被果蝇Vnd抑制的背-腹侧模式基因。这是迄今为止Nkx2.2/Vnd家族中唯一被充分表征的靶标。果蝇作为一种遗传模型，其简单而复杂的特性为从胰岛素反应到衰老的发育提供了新的见解。在这里，我们提出利用微阵列分析从果蝇胚胎中分离的mrna来鉴定Nkx2.2/vnd基因家族的靶标，这些mrna在只需要激活主要（直接）靶基因时短暂过表达vnd。为了评估这个试点项目的可行性，我们将首先关注那些已经被其他实验室研究过的候选基因和目标基因。对野生型、vnd负型和vnd过表达突变胚胎的rna进行定量RT-PCR和原位杂交，将进一步确定候选vnd靶基因是否是真正的靶标。为了确定我们鉴定的靶蝇基因的脊椎动物同源物是否对胰腺发育至关重要，我们将评估野生型和NKx2.2突变体发育中的胰腺中保守的脊椎动物同源物的表达。这项初步研究旨在利用多功能果蝇模型来获得对NKx2.2靶基因的开创性见解。这些知识可能对定义β胰岛规范所需的调节网络具有高度的指导意义。

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.