Regulation of Apical-Basal Cell polarity during Intestinal Epithelial Morphogenes

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。我之前的博士和博士后培训经验一直与肠道内胚层发育有关。我的近期目标是在未来3年内完成向胃肠道发育和疾病领域的独立科学家的过渡。我的长期目标是研究细胞极性调节及其在胃肠道形态发生和相关疾病中的作用。我在一个非常活跃的研究环境中工作，有来自遗传学系、胃肠病分部和糖尿病、肥胖和代谢疾病研究所的高度互动的科学家。克劳斯·凯斯特纳博士将是我的主要导师，而消化科科长阿尼尔·鲁斯特吉博士将是我的共同导师。凯斯特纳博士是老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道发育和疾病专家。这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奖获得者转变为独立学院的记录。其他主要顾问包括CDC42和细胞极性研究专家Erfei Bii博士，斑马鱼肠道发育专家Michael Pack博士，以及原代组织培养和器官类型培养模型专家Hiroshi Nakagawa博士。两位技术顾问乔纳森·舒格和雷蒙德·米德将就统计数据分析和EM实验向我提供建议。哺乳动物的肠道包含一个高度极化的上皮，它是消化和吸收营养的中心。虽然我们对典型的Wnt、Notch和其他信号通路在肠上皮形态发生中的作用有了足够的了解，但对关键的极性调节因子在这一过程中的作用却知之甚少。一些细胞极性基因与胃肠道疾病，特别是结肠癌有关。我最近与凯斯特纳博士的合作确立了cdx2作为肠道细胞命运的关键指导者。这项工作已经被发育细胞接受。我们对肠上皮特异性CDX2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方面发挥重要作用，并且这种作用似乎与顶端PAR复合体和CDC42极性通路有关。切除分化的肠上皮中的CDX2会导致顶端-基底侧细胞取向的破坏，形成额外的紧密连接，并损害基底膜的完整性。我们假设顶端极性复合体(Par3/Par6/aPKC/CDc42)结合了肠道细胞谱系信息，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道极化过程中PAR/aPKC和CDC42的时空活性，并探讨CDX2在形态发生过程中调节心尖极激活的分子机制。我们将使用一种新的肠道器官型培养模型，结合肠道特异的CDC42基因敲除小鼠模型，进一步确定细胞极性的主要调节因子CDC42在肠道发育中的作用。这些研究将有助于阐明哺乳动物上皮细胞形态发生过程中细胞极性调控的基本机制，有助于更好地了解相关胃肠道疾病。这项提议的目标与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，以及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器。这个奖项将保护我创造新的遗传模型的时间，包括肠道特异的CDc42基因敲除小鼠。

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.