Molecular genetics of epithelium formation in C. elegans

描述（由申请人提供）：本提案的主要目标是了解蛔虫秀丽隐杆线虫的上皮如何形成。上皮结构在身体中发挥着许多必要的作用，如作为屏障（如皮肤）或形成运输小管（如肠道、血管等）。为了实现这些不同的功能，上皮细胞必须高度极化，具有不同的膜结构域。上皮极性的调节是非常重要的，因为失调会导致人类疾病。例如，Wilms' Tumor，一种儿童癌症，可能是由Wilms' Tumor 1 （WT1）基因突变引起的。由于上皮形成缺陷，WT1突变小鼠不能形成完整的肾脏。此外，上皮极性的丧失可能有助于癌症转移。因此，确定调控上皮极化的基因是至关重要的。目前的观点是，粘附蛋白e -钙粘蛋白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，已经发现了一种不依赖钙粘蛋白的启动上皮极化的途径。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导的情况下使单个人肠细胞极化。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。为了阐明这种不依赖钙粘蛋白的途径的机制，Mango实验室进行了遗传筛选，以发现不能产生前肠上皮的突变体。令人惊讶的是，这个筛选发现了细胞分裂的调节因子，中心纺锤体成分ZEN-4/kinesin和CYK-4/GAP。前肠上皮不能形成是由于极性缺陷而不是细胞质分裂。芒果实验室的进一步研究表明，CYK-4调节RhoA活性和肌动球蛋白收缩力，从而启动秀丽隐杆线虫合子的极化。本研究旨在1)验证一个有趣的假设，即前肠上皮的形成需要与单细胞胚胎极化相同的基因；2)进行全基因组RNA干扰（RNAi）筛选，以确定调节上皮形成的新基因。在第一个目标中，我将使用荧光标记蛋白和显微镜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，以生成上皮形成的遗传途径。在第二个目标中，无偏倚的全基因组RNAi筛选新的上皮极化调节因子将涉及喂养表达dsrna的细菌以敲除基因表达。我们将详细分析阳性候选者，以发现它们在建立极性中的作用。这些结果将增加我们对这一基本细胞生物学过程的认识，并可能确定人类疾病的治疗靶点。

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.