Apical-Basal Cell polarity during Intestinal Epithelial Morphogenesis

描述（由申请人提供）： 我目前是宾夕法尼亚大学遗传学系克劳斯·凯斯特纳博士实验室的博士后研究员。我以前的博士学位。和博士后培训经验一直与肠道内胚层发育有关。在接下来的3年里，我的近期目标是完成向胃肠道发育和疾病领域独立科学家的转变。我的长期目标是研究细胞极性调节因子及其在胃肠道形态发生和相关疾病中的作用。我在一个非常动态的研究环境中工作，来自遗传学系，胃肠病学系和糖尿病，肥胖和代谢疾病研究所的科学家高度互动。Klaus Kaestner博士将是我的主要导师，Anil Rustgi博士，胃肠科主任，将是我的共同导师。Kaestner博士是小鼠遗传学和肠道发育方面的专家。Rustgi博士是一位备受尊敬的胃肠发育和疾病专家。两位导师在培训年轻科学家方面都有丰富的经验，并有成功指导和将K获奖者转变为独立学院的记录。其他主要顾问包括Cdc 42和细胞极性研究专家Erfei Bi博士，斑马鱼肠道发育专家Michael Pack博士和原代组织培养和器官型培养模型专家Hiroshi Nakagawa博士。两位技术顾问，乔纳森·舒格博士和雷蒙德·米德博士，将在统计数据分析和EM实验方面为我提供建议。哺乳动物的肠包含高度极化的上皮，其是营养物的消化和吸收的中心。虽然我们对肠上皮形态发生中的经典Wnt、Notch和其他信号通路有了充分的了解，但对关键极性调节剂在这一过程中的作用知之甚少。一些细胞极性基因与胃肠道疾病，特别是结肠癌有关。我最近与Kaestner博士的合作已经确定Cdx 2是重要的肠细胞命运主管。这项工作已被Developmental Cell接受。我们对肠上皮特异性Cdx 2基因敲除小鼠的初步数据表明，该因子在调节分化的肠上皮细胞极性形成中起重要作用，并且这种作用似乎与顶端Par复合物和Cdc 42极性通路相关。从分化的肠上皮细胞中切除Cdx 2会导致顶端-基底外侧细胞取向被破坏，形成额外的紧密连接，以及基底膜完整性受损。我们假设，顶端极性复合物（Par 3/Par 6/aPKC/Cdc 42）纳入肠细胞谱系信息，以指导上皮形态发生过程中的极性形成。我们建议分析正常肠极化过程中Par/aPKC和Cdc 42的时空活性，并研究Cdx 2在形态发生过程中调节顶端极性激活的分子机制。我们将进一步定义Cdc 42的作用，细胞极性的主调节器，在肠道的发展，使用一种新的肠道器官型培养模型，结合精氨酸特异性Cdc 42基因敲除小鼠模型。这些研究将阐明哺乳动物上皮细胞形态发生中细胞极性调控的基本机制，并有助于更好地理解相关的胃肠道疾病。本提案的目标与NIDDK的使命直接相关。培训过程将扩大我的技术能力，如开发一种新的肠道器官型培养模型，并利用免疫电子显微镜分析在肠道形态发生过程中定位极性调节剂。该奖项将保护我的时间，以产生新的遗传模型，包括尼古丁特异性Cdc 42敲除小鼠。

DESCRIPTION (provided by applicant): I am currently a postdoctoral fellow in Dr. Klaus Kaestner's laboratory in the Department of Genetics, at University of Pennsylvania. My previous Ph.D. and postdoctoral training experience has been continuously related to gut endoderm development. My immediate goal during the next 3 years is to complete the transition to an independent scientist in the field of gastrointestinal development and diseases. My long-term goal is to investigate the cell polarity regulators and their roles in the gastrointestinal morphogenesis and related diseases. I am working in a very dynamic research environment that has highly interactive scientists from the Department of Genetics, the Gastroenterology Division and the Institute for Diabetes, Obesity and Metabolic Diseases. Dr. Klaus Kaestner will be my primary mentor, and Dr. Anil Rustgi, the Chief of Gastroenterology Division, will be my co-mentor. Dr. Kaestner is an expert in mouse genetics and gut development. Dr. Rustgi is a well-respected expert in gastrointestinal development and diseases. Both mentors have ample experiences in training young scientists and have a track record of successful mentoring and transitioning K awardees into independent faculties. Other key advisors include Dr. Erfei Bi, an expert in Cdc42 and cell polarity research, Dr. Michael Pack, an expert of Zebrafish gut development, and Dr. Hiroshi Nakagawa, an expert in primary tissue culture and organotypic culture model. Two technical consultants, Drs Jonathan Schug and Raymond Meade, will advise me on statistical data analysis and EM experiments. The mammalian intestine contains a highly polarized epithelium that is central to digestion and absorption of nutrients. Although we have ample knowledge about canonical Wnt, Notch and other signaling pathways in the intestinal epithelial morphogenesis, little is known about the roles of key polarity regulators in this process. Several cell polarity genes have been implicated in gastrointestinal diseases, in particular the colon cancer. My recent work with Dr. Kaestner has established Cdx2 as the essential intestinal cell fate director. This work has been accepted by Developmental Cell. Our preliminary data on intestinal epithelium-specific Cdx2 knockout mice suggest that this factor plays an important role in regulating cell polarity formation in differentiated intestinal epithelium, and this effect appears to be associated with the apical Par complex and Cdc42 polarity pathways. Ablation of Cdx2 from differentiated intestinal epithelium leads to a disrupted apical-basolateral cell orientation, an extra tight junction formation, and an impaired basement membrane integrity. We hypothesize that apical polarity complex (Par3/Par6/aPKc/Cdc42) incorporates intestinal cell lineage information to instruct polarity formation during epithelial morphogenesis. We propose to analyze the spatial and temporal activity of Par/aPKC and Cdc42 during normal intestinal polarization, and investigate the molecular mechanism by which Cdx2 regulates apical polarity activation during the morphogenetic process. We will further define the role of Cdc42, a master regulator of cell polarity, in the development of intestine, using a novel intestinal organotypic culture model, in combination with the intestine-specific Cdc42 knockout mouse model. These studies will elucidate the basic mechanisms of cell polarity regulation in mammalian epithelial morphogenesis, and will contribute to a better understanding of related gastrointestinal diseases. The goal of this proposal is directly relevant to the mission of NIDDK. The training process will expand my technical capacities such as the development of a novel intestinal organotypic culture model, and the utilization of immuno-electron microscopic analysis to localize polarity regulators during intestinal morphogenesis. The award will protect my time for generating new genetic models including the intestine-specific Cdc42 knockout mice.