The role of TOR in digestive tract development

TOR 在消化道发育中的作用

• 批准号: 7229863
• 负责人: ALAN Neil MAYER
• 金额: $ 14.71万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-10 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7229863
• 关键词: Address; Amino Acyl-tRNA Synthetases; Biogenesis; CCI-779; Cells; Defect; Development; Disease; Embryo; Embryonic Development; End Point; Enzymes; Failure; Functional Imaging; Future; Gastrointestinal tract structure; Genes; Genetic Screening; Growth Factor; Homeostasis; Intestines; Intravenous; Life; Measures; Mediating; Metabolic; Methods; Molecular; Monitor; Morbidity - disease rate; Mutation; Nutrient; Organ; Organ Specificity; Organogenesis; Ornithine Decarboxylase; PTEN gene; Pathway interactions; Peptide Initiation Factors; Phenotype; Phosphotransferases; Play; Primordium; Protein Biosynthesis; Proteins; RNA Processing; Regulation; Reporter; Research; Research Personnel; Resolution; Rewards; Ribosomal Proteins; Risk; Role; Signal Pathway; Signal Transduction; Sirolimus; Small Intestines; Specificity; System; TSC1/2 gene; Testing; Time; Transgenic Organisms; Translations; Transplantation; Validation; Variant; Zebrafish; analog; base; cell growth; fluorescence imaging; in vivo; mortality; novel; nutrition; organ regeneration; programs; red fluorescent protein; tool; transmission process

DESCRIPTION (provided by applicant): Understanding intestinal development at the molecular level is critical for developing future methods of promoting organ regeneration and treatments for intestinal failure. Genetic screens in the zebrafish have identified numerous genes required for intestinal development that are also known to be involved in protein biosynthesis. A common feature of many of these protein biosynthesis genes is their regulation by the target of rapamycin (TOR). That mutations in genes controlled by TOR are selective to the digestive organs suggests that an additional common mechanism of control exists that provides organ specificity to the phenotype of these defects. It is our hypothesis that TOR is the leading candidate for mediating this specificity given its central role in regulating these anabolic pathways and their gene products. Testing this hypothesis in a future R01 proposal will require addressing experimentally several important questions: Does TOR activity vary in time and space during development? Does TOR activity correlate with particular morphological transitions? Are certain organs are more reliant on TOR activity than others? Are there subsets of cells in developing organs with high levels of TOR activity? Most importantly, what is the functional significance of TOR signaling during development? At present, there is no experimental system for addressing these questions. Accordingly, in this R21 exploratory proposal we aim to develop a novel tool we term the "TORfish" that will allow us to address these questions experimentally by permitting the functional imaging of TOR activity in living vertebrate embryos. Specific Aim 1: We will create a novel transgenic zebrafish reporter line for functional imaging of TOR signaling activity in vivo (TORfish): In particular, we will generate dicistronic constructs containing a TOR-dependent reporter fused to a TOR-independent reporter to normalize for transcriptional variation, screen reporter constructs for rapamycin sensitive translation; and create transgenic lines with germline transmission of the reporter construct. Specific Aim 2: We will validate the TORfish and assess the role of TOR signaling during digestive organ development using the TORfish: In particular, we will (a) perform time-lapse fluorescence imaging of TORfish embryos during normal development, (b) phamacologically inhibit TOR with rapamycin or its analogs CCI-779 (Wyeth) and RAD-001 (Novartis), and (c) employ antisense morpholinos to knockdown TOR, its positive downstream effectors (p70S6 kinase, eIF4E), and its antagonists (PTEN, TSC1/TSC2, and 4E-BP). Successful development and validation of the TORfish will allow us to experimentally test our hypothesis that TOR signaling plays an important role in organ-specific organogenesis in a future RO1 proposal. The development of the TORfish in this R21 exploratory proposal therefore represents an important risk-reward hurdle that must be overcome in order to pursue future hypothesis-driven research in this system.

描述（由申请人提供）：在分子水平上了解肠道发育对于开发未来促进器官再生和治疗肠衰竭的方法至关重要。斑马鱼的遗传筛选已经确定了肠道发育所需的许多基因，这些基因也已知参与蛋白质生物合成。许多这些蛋白质生物合成基因的共同特征是它们受雷帕霉素（TOR）靶点的调节。由TOR控制的基因突变对消化器官具有选择性，这表明存在另一种共同的控制机制，为这些缺陷的表型提供器官特异性。我们的假设是，TOR是介导这种特异性的主要候选者，因为它在调节这些合成代谢途径及其基因产物中起着核心作用。 在未来的R 01提案中验证这一假设将需要通过实验解决几个重要问题：TOR活动在发育过程中是否随时间和空间而变化？TOR活性是否与特定的形态学转变相关？某些器官是否比其他器官更依赖于TOR活动？在发育中的器官中是否存在具有高水平TOR活性的细胞亚群？最重要的是，TOR信号在发育过程中的功能意义是什么？目前，还没有解决这些问题的实验系统。因此，在这个R21探索性提案中，我们的目标是开发一种新的工具，我们称之为“TORFish”，这将使我们能够通过允许活脊椎动物胚胎中TOR活性的功能成像来解决这些问题。 具体目标1：我们将创建一种新的转基因斑马鱼报告细胞系，用于体内TOR信号传导活性的功能成像（TORFish）：特别是，我们将生成含有融合至TOR非依赖性报告细胞的TOR依赖性报告细胞的双顺反子构建体，以标准化转录变异，筛选雷帕霉素敏感性翻译的报告细胞构建体;并创建具有报告细胞构建体的种系传递的转基因细胞系。 具体目标二：我们将验证TORFish，并使用TORFish评估TOR信号在消化器官发育过程中的作用：特别地，我们将（a）在正常发育期间对TOR鱼胚胎进行延时荧光成像，（B）用雷帕霉素或其类似物CCI-779（Wyeth）和RAD-001药理学地抑制TOR（Novartis），和（c）使用反义吗啉代敲低TOR、其阳性下游效应物（p70 S6激酶，eIF 4 E）及其拮抗剂（PTEN，TSC 1/TSC 2和4 E-BP）。TORFish的成功开发和验证将使我们能够通过实验验证我们的假设，即TOR信号在未来的RO 1提案中在器官特异性器官发生中起着重要作用。因此，在R21探索性提案中，TORFish的开发代表了一个重要的风险-回报障碍，必须克服这个障碍，以便在该系统中进行未来的假设驱动研究。