The Division and Homeostasis of Gut Progenitor Cells

描述（由申请人提供）：在理解如何在哺乳动物上皮细胞中控制Wnt蛋白的高尔基体后转运和分泌以维持组织稳态方面存在根本性的差距。这种知识差距的持续存在阻碍了对Wnt生物学及其对结肠直肠癌等人类疾病的贡献的全面理解。长期目标是阐明关键细胞内运输机制的分子机制，该机制部署和协调对以下过程至关重要的信号事件： 造血干细胞更新与上皮细胞稳态。本申请的目的是确定Rab 8和Rab 11调节的胞吐和再循环内体区室在肠隐窝稳态和干细胞分裂中的分子功能。核心假设是Rab 8和Rab 11介导的囊泡出芽和运输事件执行Wnt/Wntless的高尔基体后运输到质膜进行分泌。这一假设是根据申请人实验室产生的初步数据提出的。拟议研究的基本原理是，了解细胞内Wnt转运和分泌的调控机制将产生对操纵Wnt产生的新策略的翻译见解。在强有力的体外和体内初步数据的指导下，该假设将通过追求两个特定目标进行测试：（1）确定Rab 8a在Wnt分泌和隐窝稳态中的作用;（2）确定Rab 11 a对Wnt分泌和隐窝稳态的贡献。我们将在第一个目标中确定Rab 8囊泡运输机制在小鼠肠道和原代细胞系中对Wnt转运和释放的调节。Rab 11 a缺陷对肠上皮形态发生和干细胞分裂的影响将在第二个目标下确定。将使用现有的和新衍生的敲除小鼠系，以及现有的基因工程原代小鼠胚胎细胞系。该提议是创新性的，因为（1）它利用了新的敲除小鼠模型和细胞系，这代表了对研究这些小GTP酶的现有方法的实质性偏离;以及（2）它探索了体内Wnt分泌的调节，这在概念上是针对Wnt下游信号传导事件的研究的新颖之处。这个项目是重要的，因为它是一个连续的研究，以更好地了解Wnt配体的释放和接收在干细胞和癌症生物学的关键一步。这些知识最终将转化为用于治疗Wnt相关人类疾病的改进的治疗策略。此外，从小鼠肠上皮细胞获得的见解将对其他哺乳动物组织和疾病中形态梯度形成和维持的机制产生更广泛的影响。 公共卫生相关性：该项目使用基因敲除小鼠模型和基因工程细胞系来探索Wnt运输和分泌的调节。细胞分裂和 小肠隐窝室的内环境稳定将是这项资助的主要重点。

DESCRIPTION (provided by applicant): There is a fundamental gap in understanding how the post-Golgi transport and secretion of Wnt proteins are controlled in mammalian epithelial cells in order to sustain tissue homeostasis. Continued existence of this knowledge gap prevents the complete understanding of Wnt biology and its contribution to human diseases such as colorectal cancers. The long-term goal is to elucidate the molecular mechanisms of key intracellular trafficking machinery that deploys and coordinates the signaling events essential for intesti- nal stem cell renewal and epithelial homeostasis. The objective of this application is to determine the molecular function of Rab8- and Rab11-regulated exocytotic and recycling endosome compartments in intestinal crypt homeostasis and stem cell division. The central hypothesis is that Rab8- and Rab11- mediated vesicle budding and trafficking events execute the post-Golgi transport of Wnt/Wntless to plasma membrane for secretion. This hypothesis has been formulated on the basis of preliminary data generated in the applicant's laboratory. The rationale for the proposed research is that understanding the regulatory mechanisms of intracellular Wnt transport and secretion will yield translational insights into novel strategies fr manipulating Wnt production. Guided by strong in vitro and in vivo preliminary data, this hypothesis will be tested by pursuing two specific aims: (1) To define the role of Rab8a in Wnt secre- tion and crypt homeostasis; and (2) To identify the contribution of Rab11a to Wnt secretion and crypt homeostasis. We will determine the regulation of Wnt transport and release by Rab8 vesicle trafficking machinery in mouse intestines and primary cell lines in the first aim. The impact of Rab11a deficiency on intestinal epithelial morphogenesis and stem cell division will be determined under the second aim. Exist- ing and newly-derived knockout mouse lines, as well as genetically engineered primary mouse embry- onic cell lines that are already on hand will be used. This proposal is innovative, because (1) it utilizes new knockout mouse model and cel lines, which represent a substantive departure from the existing methodologies for studying these small GTPases; and (2) it explores the regulation of Wnt secretion in vivo, which is conceptually novel from studies focused on Wnt downstream signaling events. This project is significant because it is a critical step in a continuum of research towards the better understanding of Wnt ligand release and reception in stem cell and cancer biology. Such knowledge will ultimately trans- late into improved therapeutic strategies for the treatment of Wnt-related human diseases. In addition, insights gained from mouse intestinal epithelium will have a broader impact on the mechanism of the morphogen gradient formation and maintenance in other mammalian tissues and diseases. PUBLIC HEALTH RELEVANCE: This project uses knockout mouse models and genetically engineered cell lines to explore the regulation of Wnt traffic and secretion. The cell division and homeostasis of the smal intestinal crypt compartment will be the major focus of this grant.