Molecular genetics of epithelium formation in C. elegans

线虫上皮形成的分子遗传学

• 批准号: 7879908
• 负责人: Stephen E Von Stetina
• 金额: $ 5.38万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7879908
• 关键词: Actomyosin; Adhesions; Antibodies; Apical; Applications Grants; Bacteria; Biological; Biological Process; Blood Vessels; Cadherins; Caenorhabditis elegans; Cell Polarity; Cells; Cues; Cytokinesis; Defect; Development; Double-Stranded RNA; E-Cadherin; Embryo; Embryonic Development; Epidermis; Epithelial; Epithelial Cells; Epithelium; Event; Failure; Foundations; Gene Expression; Generations; Genes; Genetic; Genetic Epistasis; Genetic Screening; Goals; Hand; Human; Imaging Techniques; Intestines; Kidney; Kinesin; Knowledge; Link; Maintenance; Malignant Childhood Neoplasm; Mango - dietary; Mediating; Membrane; Mesenchymal; Microscopy; Mitotic; Modeling; Molecular Genetics; Monomeric GTP-Binding Proteins; Mutant Strains Mice; Mutation; Nematoda; Neoplasm Metastasis; Nephroblastoma; Oral cavity; Organ; Organism; Organogenesis; Pathway interactions; Pharyngeal structure; Primitive foregut structure; Process; Protein-Serine-Threonine Kinases; Proteins; RNA Interference; Reagent; Regulation; Reporter; Role; STK11 gene; Screening procedure; Signal Induction; Signal Transduction; Skin; Structure; System; Testing; Tube; Vertebrates; Visual; WT1 gene; Work; alpha catenin; base; beta catenin; embryo cell; feeding; genome-wide; human disease; knock-down; mutant; novel; polarized cell; protein E; research study; rho GTPase-activating protein; spindlin; therapeutic target; zygote

DESCRIPTION (provided by applicant): The main goal of this proposal is to understand how epithelia are formed in the roundworm C. elegans. Epithelial structures perform many necessary roles in the body, such as acting as barriers (e.g.: skin) or forming transport tubules (e.g. gut, blood vessels, etc.). To execute these diverse functions, epithelial cells must be highly polarized with distinct membrane domains. The regulation of epithelial polarity is of high importance, as dysregulation can result in human disease. For example, Wilms' Tumor, a childhood cancer, can be caused by a mutation in the Wilms' Tumor 1 (WT1) gene. WT1 mutant mice fail to form a complete kidney due to a defect in epithelium formation. In addition, the loss of epithelial polarity may contribute to cancer metastasis. Thus, it is crucial to determine the genes that regulate epithelial polarization. The current view is that the adhesion protein E-cadherin is necessary to initiate and maintain epithelial polarity. However, a cadherin-independent pathway that initiates epithelial polarization has been uncovered in many systems, including vertebrates. For example, the serine/threonine kinase LKB1 is sufficient to polarize single human intestine cells in the absence of cadherin-mediated signaling. In addition, C. elegans mutants lacking a functional cadherin still have polarized epithelia. To elucidate the mechanism of this cadherin-independent pathway, the Mango lab performed a genetic screen to discover mutants that fail to generate a foregut epithelium. Surprisingly, this screen identified regulators of cytokinesis, the central-spindlin components ZEN-4/kinesin and CYK-4/GAP. The failure to form a foregut epithelium resulted from a defect in polarity and not in cytokinesis. Further work from the Mango lab showed that CYK-4 regulates RhoA activity and actomyosin contractility to initiate polarization of the C. elegans zygote. This proposal aims to 1) Test the intriguing hypothesis that formation of a foregut epithelium requires the same genes that act to polarize the one-cell embryo and 2) Perform a genome-wide RNA interference (RNAi) screen to identify new genes regulating epithelium formation. In the first aim I will use fluorescently-tagged proteins and microscopy to document carefully the events during polarization of the foregut epithelium. I will also determine at which step ZEN-4 and CYK-4 act, and perform mutant analysis to generate a genetic pathway for epithelium formation. In the second aim, the unbiased, genome-wide RNAi screen for novel regulators of epithelial polarization will involve feeding dsRNA-expressing bacteria to knock-down gene expression. Positive candidates will be analyzed in detail to discover their role in establishing polarity. These results will add to our knowledge of this basic cell biological process and may identify therapeutic targets for human disease.

描述（由申请人提供）：该提案的主要目标是了解如何在round虫秀丽隐杆线虫中形成上皮。上皮结构在体内发挥许多必要的作用，例如作用为屏障（例如，皮肤）或形成运输小管（例如肠道，血管等）。为了执行这些不同的功能，上皮细胞必须具有不同的膜结构域高度极化。上皮极性的调节非常重要，因为失调会导致人类疾病。例如，Wilms的肿瘤是一种儿童癌，可能是由Wilms肿瘤1（WT1）基因突变引起的。由于上皮形成缺陷，WT1突变小鼠无法形成完整的肾脏。另外，上皮极性的丧失可能导致癌症转移。因此，确定调节上皮极化的基因至关重要。当前的观点是，粘附蛋白E-钙粘蛋白对于启动和维持上皮极性是必要的。然而，在包括脊椎动物在内的许多系统中发现了启动上皮极化的cadherin独立途径。例如，在没有钙粘蛋白介导的信号传导的情况下，丝氨酸/苏氨酸激酶LKB1足以使单个人肠细胞两极化。此外，缺乏功能性钙粘蛋白的秀丽隐杆线虫突变体仍然具有极化上皮。为了阐明这种非核蛋白独立途径的机制，芒果实验室进行了遗传筛选，以发现未能产生前肢上皮的突变体。令人惊讶的是，该屏幕确定了细胞因子的调节剂，中央Spindlin组件ZEN-4/驱动蛋白和CYK-4/GAP。未能形成前肢上皮是由于极性缺陷而不是细胞因子的缺陷。芒果实验室的进一步工作表明，CYK-4调节RhoA活性和肌动球蛋白的收缩力，以启动秀丽隐杆线虫的极化。该提议的目的是1）检验以下有趣的假设，即前述上皮的形成需要相同的基因，以使单细胞胚胎两极化和2）进行全基因组RNA干扰（RNAI）筛查以识别调节上皮形成的新基因。在第一个目的中，我将使用荧光标记的蛋白质和显微镜仔细记录上述上皮极化期间的事件。我还将在哪个步骤ZEN-4和CYK-4 ACT上确定突变分析以产生上皮形成的遗传途径。在第二个目标中，用于上皮极化的新调节剂的无偏，全基因组的RNAi筛选将涉及喂养表达DSRNA的细菌以敲除基因表达。将对积极的候选人进行详细分析，以发现其在建立极性中的作用。这些结果将增加我们对这个基本细胞生物学过程的了解，并可能确定人类疾病的治疗靶标。