Genetic Control of Intestinal Epithelial Stem Cell Maintenance & Differentiation

描述(申请人提供)：位于隐窝底部的多能肠上皮干细胞(IESCs)池不断更新肠上皮。IESCs的定义是a)自我更新的能力，以及b)产生所有终末分化的肠道上皮细胞谱系的多能性--也就是众所周知的“干性”特征。IESC生物学中的一个主要问题是什么控制茎。Sox(Sry Box)转录因子家族的成员在其他组织中作为茎的主控开关发挥作用。这项建议旨在确定Sox9在IESC茎干中的特定作用和基因靶标。我们最近证明，不同水平的Sox9可以不同地标记功能性IESCs、祖细胞、肠内分泌细胞和Paneth细胞。Sox9和Lgr5都标记了介于Paneth细胞之间并与Paneth细胞密切接触的IESCs。新的证据表明，Paneth细胞起着“护士”细胞的作用，对IESC的维持至关重要。在胚胎发育期间，切除肠上皮中的Sox9会导致Paneth细胞的丢失，并增加小肠和结肠隐窝内的增殖。我们关于可诱导的Sox9消融成人肠上皮短期效应的新的初步数据证实了隐窝的过度增殖，但也为其他表型后果提供了新的证据，包括Paneth细胞的扩张和移位、Paneth细胞的形态缺陷、杯状细胞生物标记物Muc2在Paneth细胞中的错误表达、基于隐窝的肠内分泌细胞和杯状细胞的减少，以及上皮单层特征的丧失。对正常小肠和可诱导条件Sox9突变的小肠的基因芯片分析表明，Sox9控制着与细胞增殖、细胞黏附和迁移相关的基因和途径。由于我们的初步研究以泛上皮方式去除Sox9，他们没有区分IESC中Sox9的缺失与其他表达Sox9的细胞系(包括Paneth细胞)相比，哪些表型效应是由Sox9的缺失引起的。中心假说是，Sox9通常通过a)对IESCs内特定靶基因的内在调控，以及b)通过旁分泌影响或通过将Paneth细胞限制在IESC小生境，从外部控制IESC增殖或潜能的Paneth细胞特异性基因，来限制IESC的增殖并保持多能性。为了验证这一假说，我们提出了以下目标：1)确定Sox9在IESCs中的特定作用，2)确定Sox9调控的基因如何影响IESC的干细胞性，3)确定Sox9在a)IESCs和b)Paneth细胞中调控的特定基因靶点和网络。这些研究的成功完成将带来新的发现，包括控制IESC行为的内在和外在机制。这些发现可能会延伸到与其他组织特异性干细胞相关的平行领域。 公共卫生相关性：肠上皮干细胞(IESCs)不断地再生小肠和结肠内的细胞，在肠道内稳态和疾病中发挥重要作用。了解控制IESCs的分子过程对于开发下一代治疗肠道疾病的方法至关重要。拟议的项目将导致对IESCs的一种非常有吸引力的分子调节因子-转录因子Sox9的全面研究。我们预测，这是从细胞内和通过邻近细胞控制IESC的行为。为了测试这一点，将产生并表征在IESCs中缺乏或过度表达Sox9的小鼠。这些研究将展示Sox9的干细胞特异性作用，并展示该基因如何内在地调节IESCs。先进的分子生物学技术将被用来识别Sox9与之相互作用的基因，使研究团队能够清楚地了解Sox9参与的调控网络。这将有助于更好地理解IESCs的基因控制。此外，在Paneth细胞中缺乏Sox9的小鼠被认为是IESCs的“哺育细胞”，将因其对IESC干细胞的影响而被表征。这一目标将确定Sox9在调控IESCs中扮演的外部角色，这是一种在干细胞生物学概念上新颖的方法。总体而言，这笔赠款中建议的项目将通过研究强大的调控基因，使人们对IESC的基本生物学有更详细的了解，并促进开发针对小肠和结肠疾病的新疗法所需的知识。

DESCRIPTION (provided by applicant): The intestinal epithelium is constantly renewed by a pool of multipotent intestinal epithelial stem cells (IESCs) located at the crypt base. IESCs are defined by their a) competence to self-renew, and b) multipotency to give rise to all terminally differentiated intestinal epithelial lineages - characteristics otherwise known as 'stemness'. A major question in IESC biology is what controls stemness. Members of the Sox (Sry Box) family of transcription factors function as master control switches for stemness in other tissues. This proposal aims to define the specific roles and gene targets of Sox9 in IESC stemness. We recently demonstrated that distinct levels of Sox9 differentially mark functional IESCs, progenitors, enteroendocrine cells and Paneth cells. Sox9 and Lgr5 both mark IESCs that are intercalated between and make intimate contact with Paneth cells. New evidence suggests that Paneth cells serve as 'nurse' cells and are critical for IESC maintenance. Ablation of Sox9 in intestinal epithelium during embryogenesis results in the loss of Paneth cells and increased proliferation within the crypts of the small intestine and colon. Our new preliminary data on short-term effects of inducible Sox9 ablation in the adult intestinal epithelium confirm crypt hyperproliferation, but also provide novel evidence for additional phenotypic consequences including expansion and displacement of Paneth cels, morphological defects in Paneth cells, mis-expression of goblet biomarker Muc2 in Paneth cells, reductions in crypt-based enteroendocrine cells and goblet cells, and loss of epithelial mono-layer characteristics. Gene microarray on normal small intestine versus intestine from inducible conditional Sox9-mutants indicates that Sox9 controls genes and pathways involved in proliferation, cell adhesion and migration. Since our preliminary studies ablated Sox9 in a pan-epithelial manner, they do not distinguish which phenotypic effects result from the loss of Sox9 in the IESC versus other Sox9-expressing cell lineages, including Paneth cells. The central hypothesis is that Sox9 normally limits IESC proliferation and preserves multipotency by a) intrinsic regulation of specific target genes within IESCs, and b) regulation of Paneth cell specific genes that extrinsically control IESC proliferation or potency by paracrine influences or by restricting Paneth cells to the IESC niche. To test this hypothesis we propose the following aims: 1) to determine the specific roles of Sox9 in IESCs, 2) to determine how Sox9 regulated genes in Paneth cells impacts IESC stemness, 3) to identify specific gene targets and networks regulated by Sox9 in a) IESCs and b) Paneth cells. Successful completion of these studies will lead to novel findings including intrinsic and extrinsic mechanisms controlling IESC behavior. These findings will likely extend to parallel fields of study related to other tissue specific stem cells. PUBLIC HEALTH RELEVANCE: Intestinal epithelial stem cells (IESCs) constantly regenerate the cells lining the small intestine and colon and play an important role in intestinal homeostasis and disease. Understanding the molecular processes that control IESCs is critical to developing the next generation of therapies against intestinal disease. The proposed project will result in the comprehensive study of a very attractive molecular regulator of IESCs, the transcription factor Sox9. We predict that is controlling IESC behavior from within the cell and through neighboring cells. To test this, mice that are deficient for or over- express Sox9 specifically in IESCs will be generated and characterized. These studies will demonstrate the stem cell-specific roles of Sox9 and demonstrate how the gene regulates IESCs intrinsically. Advanced molecular biology techniques will be utilized to identify genes that Sox9 interacts with, allowing the research team to develop a clear picture of the regulatory network that Sox9 participates in. This will lead to a better understanding of genetic control of IESCs. Additionally, mice lacking Sox9 in Paneth cells, which have been proposed to be "nurse cells" for IESCs, will be characterized for their impact on IESC stemness. This aim will identify extrinsic roles Sox9 plays in regulating IESCs, an approach that is conceptually novel to stem cell biology. As a whole, the projects proposed in this grant will result in a more detailed understanding of basic IESC biology through the study of a powerful regulatory gene and advance the knowledge necessary to develop new therapies against disease of the small intestine and colon.