Pancreas-specific primary regulatory targets of Nkx2.2

Nkx2.2 胰腺特异性主要调控靶标

• 批准号: 6675089
• 负责人: DERVLA M MELLERICK-DRESSLER
• 金额: $ 15.3万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6675089
• 关键词: Drosophilidae; animal tissue; arthropod genetics; cell differentiation; developmental genetics; functional /structural genomics; gene expression; genetic models; genetically modified animals; high throughput technology; homeobox genes; in situ hybridization; messenger RNA; microarray technology; pancreas; pancreatic islets; polymerase chain reaction; transcription factor

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.

描述（由申请人提供）：Nkx2.2/vnd 家族的同源盒基因是胰腺规格和腹侧神经管模式化所必需的。该基因家族在不同动物物种（包括果蝇和人类）的谱系规范中发挥保守作用。尽管无脊椎动物缺乏胰腺，但神经和消化系统中的内分泌细胞会分泌胰岛素来协调细胞代谢和营养状况。最近在果蝇中描述了七个胰岛素样基因。胰腺和神经谱系具有祖先相关性，并且具有共同的调节因子。在小鼠中，胰岛素增强剂驱动腹侧神经管和胰腺的表达，而果蝇神经元则产生胰岛素。此外，暴露于产生神经元的因子的ES细胞会发育成运动神经元和胰岛。 Nkx2.2/Vnd 型转录因子对于胰腺和神经规范方面至关重要，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仍未确定。我们之前描述过，ind，一个背腹模式基因，直接被果蝇 Vnd 抑制。这是迄今为止 Nkx2.2/Vnd 系列中唯一具有明确特征的目标。果蝇作为一种简单而复杂的遗传模型，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，我们建议使用微阵列分析从果蝇胚胎中分离出的 mRNA 来识别 Nkx2.2/vnd 基因家族的靶标，这些胚胎在仅应激活主要（直接）靶基因时短暂过度表达 vnd。为了评估这个试点项目的可行性，首先我们将重点关注其他实验室之前研究过的候选 vnd 靶基因。对野生型、vnd 缺失和 vnd 过表达突变体胚胎的 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。为了确定我们确定的目标果蝇基因的脊椎动物同源物是否对胰腺发育至关重要，将评估野生型和 NKx2.2 突变体发育中胰腺中保守脊椎动物同源物的表达。该试点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。这些知识对于定义 β 胰岛规范所需的调节网络可能非常有指导意义。