The role of TOR in digestive tract development

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道衰竭的方法至关重要。斑马鱼的基因筛查已经确定了许多肠道发育所需的基因，这些基因也被认为与蛋白质的生物合成有关。许多这些蛋白质生物合成基因的一个共同特征是它们受到雷帕霉素(TOR)靶标的调控。由TOR控制的基因突变对消化器官具有选择性，这表明存在另一种常见的控制机制，为这些缺陷的表型提供了器官特异性。我们的假设是，鉴于TOR在调节这些合成代谢途径及其基因产物中的核心作用，它是调节这种特异性的主要候选者。 在未来的R01提案中验证这一假设将需要从实验上解决几个重要的问题：TOR活动在发育过程中是否会在时间和空间上发生变化？TOR活动是否与特定的形态转变相关？是不是某些器官比其他器官更依赖TOR活性？发育中的器官中是否存在高水平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R21的探索性提案中，我们的目标是开发一种新的工具，我们将其命名为“TORFish”，它将允许我们通过允许在活的脊椎动物胚胎中进行TOR活动的功能成像来实验解决这些问题。 具体目标1：我们将创建一种新型的转基因斑马鱼报告系，用于体内TOR信号活性的功能成像(TORFish)：特别是，我们将产生包含TOR依赖的报告基因的双顺反子构建体，融合到TOR非依赖的报告基因以使转录变异标准化，筛选雷帕霉素敏感翻译的报告基因构建体；并创建报告构建体的生殖系传递的转基因系。 具体目标2：我们将验证TORFISH并利用TORFISH评估TOR信号在消化器官发育中的作用：尤其是我们将(A)对TORFish正常发育期间的胚胎进行延时荧光成像，(B)用雷帕霉素或其类似物CCI-779(惠氏)和RAD-001(诺华)从药理上抑制TOR，以及(C)利用反义吗啉抑制TOR、其正向下游效应因子(p70S6激酶、eIF4E)及其拮抗剂(PTEN、TSC1/TSC2和4E-BP)。TORFISH的成功开发和验证将使我们能够在未来的RO1提案中实验验证我们的假设，即TOR信号在器官特异性器官发生中发挥重要作用。因此，在R21探索性提案中开发TORFISH代表了一个重要的风险-回报障碍，为了在该系统中进行未来的假设驱动的研究，必须克服这一障碍。

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.