Molecular genetics of epithelium formation in C. elegans

线虫上皮形成的分子遗传学

• 批准号: 7657503
• 负责人: Stephen E Von Stetina
• 金额: $ 5.17万
• 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/7657503
• 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(申请人提供)：这项建议的主要目标是了解线虫的上皮是如何形成的。上皮结构在人体中扮演着许多必要的角色，例如作为屏障(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，上皮细胞必须高度极化，具有不同的膜域。上皮极性的调节非常重要，因为调节失调会导致人类疾病。例如，Wilms‘s瘤，一种儿童癌症，可能由Wilms’s Tumor 1(WT1)基因突变引起。WT1突变小鼠由于上皮形成缺陷而无法形成完整的肾脏。此外，上皮极性的丧失可能与癌症转移有关。因此，确定调控上皮细胞极化的基因至关重要。目前的观点认为，黏附蛋白E-钙粘附素是启动和维持上皮极性所必需的。然而，在包括脊椎动物在内的许多系统中，已经发现了一种启动上皮极化的钙粘附素非依赖性途径。例如，丝氨酸/苏氨酸激酶LKB1足以在没有钙粘蛋白介导的信号传递的情况下使单个人类肠道细胞极化。此外，缺乏功能钙粘附素的线虫突变体仍然具有极化的上皮细胞。为了阐明这种钙粘素非依赖途径的机制，芒果实验室进行了基因筛查，以发现无法产生前肠上皮的突变。令人惊讶的是，这一筛选确定了胞质分裂的调节因子，中央纺锤体成分Zen-4/Kinesin和Cyk-4/GAP。未能形成前肠上皮的原因是极性缺陷，而不是胞质分裂。芒果实验室的进一步研究表明，Cyk-4调节RhoA活性和肌球蛋白收缩能力，启动线虫受精卵的极化。这项建议旨在1)检验这一有趣的假设，即前肠上皮的形成需要与单细胞胚胎极化作用相同的基因，2)进行全基因组RNA干扰(RNAi)筛选，以确定调控上皮形成的新基因。在第一个目标中，我将使用荧光标记的蛋白质和显微镜来仔细记录前肠上皮极化过程中的事件。我还将确定Zen-4和Cyk-4在哪一步起作用，并进行突变分析，以产生上皮形成的遗传途径。在第二个目标中，无偏见的、全基因组的RNAi筛选新的上皮极化调节因子将涉及喂养表达dsRNA的细菌来下调基因表达。积极的候选人将被详细分析，以发现他们在建立两极地位方面的作用。这些结果将增加我们对这一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点。