Pancreas-specific primary regulatory targets of Nkx2.2

描述(由申请人提供)：Nkx2.2/VND家族的同源框基因是胰腺规范和腹侧神经管模式所必需的。这个基因家族在包括苍蝇和人在内的不同动物物种的谱系特征中扮演着保守的角色。尽管无脊椎动物没有胰腺，但神经和消化系统中的内分泌细胞会分泌胰岛素，以协调细胞新陈代谢和营养状况。最近在果蝇中描述了七个胰岛素样基因。胰腺和神经谱系是祖先相关的，并共享共同的调节因素。在小鼠中，胰岛素增强器驱动腹神经管和胰腺中的表达，而苍蝇神经元则产生胰岛素。此外，ES细胞暴露在产生神经元的因素下，同时发育成运动神经元和胰岛。Nkx2.2/VND类转录因子对胰腺和神经的特性至关重要，但该家族如何发挥作用尚不清楚，主要是因为靶基因在很大程度上仍未确定。我们之前描述了一种背腹模式基因，该基因直接被果蝇VND抑制。这是迄今为止Nkx2.2/VND家族中唯一具有良好特征的目标。果蝇作为一种遗传模型，其简单却又复杂，为从胰岛素反应到衰老等发育方面提供了新的见解。在这里，我们建议使用从果蝇胚胎中分离的mRNAs的微阵列分析来识别Nkx2.2/vnd基因家族的靶点，这些mRNAs在只需要激活主要(直接)靶基因的时候瞬时过表达vnd。为了评估这一试点项目的可行性，我们首先将重点放在那些先前已被其他实验室研究过的候选VND靶基因上。对野生型、VND缺失和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靶基因是否为真正的靶标。为了解决我们确定的目标苍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突变体在发育中的胰腺中保守的脊椎动物同源物的表达。这项先导性研究旨在利用多功能的果蝇模型来获得对NKx2.2靶基因的开创性见解。这一知识可能对定义β胰岛规范所需的调控网络具有很大的指导意义。

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.