Genetic Control of Intestinal Epithelial Stem Cell Maintenance & Differentiation

描述（由申请人提供）：肠上皮不断更新的多能肠上皮干细胞（IESC）位于隐窝基地池。IESC由其a）自我更新的能力和B）产生所有终末分化的肠上皮谱系的多能性定义-特征也称为“干性”。IESC生物学中的一个主要问题是什么控制了干细胞。Sox（Sry Box）转录因子家族的成员在其他组织中充当干性的主控开关。本研究旨在明确Sox 9在IESC干性中的具体作用和基因靶点。我们最近证明，不同水平的Sox 9差异标记功能IESC，祖细胞，肠内分泌细胞和潘氏细胞。Sox 9和Lgr 5都标记嵌入潘氏细胞之间并与潘氏细胞密切接触的IESC。新的证据表明，潘氏细胞作为“护士”细胞，对IESC的维持至关重要。在胚胎发生期间，在肠上皮中Sox 9的消融导致潘氏细胞的损失和小肠和结肠的隐窝内的增殖增加。我们关于诱导型Sox 9消融在成人肠上皮中的短期作用的新的初步数据证实了隐窝过度增殖，但也为额外的表型后果提供了新的证据，包括潘氏细胞的扩增和移位、潘氏细胞中的形态缺陷、潘氏细胞中杯状生物标志物Muc 2的错误表达、基于隐窝的肠内分泌细胞和杯状细胞的减少，和上皮单层特征的丧失。在正常小肠与来自可诱导条件Sox 9突变体的肠的基因微阵列上表明，Sox 9控制参与增殖、细胞粘附和迁移的基因和途径。由于我们的初步研究以泛上皮的方式消融Sox 9，因此他们无法区分IESC与其他表达Sox 9的细胞系（包括Paneth细胞）中Sox 9缺失导致的表型效应。中心假设是Sox 9通常限制IESC增殖并通过以下方式保持多能性：a）IESC内特异性靶基因的内在调节，和B）通过旁分泌影响或通过将潘氏细胞限制于IESC小生境而对潘氏细胞特异性基因进行调节，所述潘氏细胞特异性基因外源性控制IESC增殖或效力。为了验证这一假设，我们提出了以下目标：1）确定Sox 9在IESC中的特定作用，2）确定Paneth细胞中Sox 9调控的基因如何影响IESC的干性，3）鉴定a）IESC和B）Paneth细胞中Sox 9调控的特定基因靶标和网络。这些研究的成功完成将导致新的发现，包括内在和外在机制控制IESC的行为。这些发现可能会扩展到与其他组织特异性干细胞相关的平行研究领域。 公共卫生相关性：肠上皮干细胞（IESC）不断再生小肠和结肠内衬细胞，并在肠道内稳态和疾病中发挥重要作用。了解控制IESC的分子过程对于开发下一代肠道疾病疗法至关重要。拟议的项目将导致一个非常有吸引力的IESCs的分子调节因子，转录因子Sox 9的全面研究。我们预测，是控制IESC的行为，从细胞内，并通过邻近的细胞。为了测试这一点，将产生并表征在IESC中特异性缺乏或过表达Sox 9的小鼠。这些研究将证明Sox 9的干细胞特异性作用，并证明该基因如何内在地调节IESC。将利用先进的分子生物学技术来识别与Sox 9相互作用的基因，使研究小组能够清楚地了解Sox 9参与的调控网络。这将有助于更好地了解IESC的遗传控制。此外，在Paneth细胞中缺乏Sox 9的小鼠（已被提出为IESC的“护士细胞”）将表征其对IESC干细胞性的影响。这一目标将确定Sox 9在调节IESC中发挥的外在作用，这是一种对干细胞生物学概念新颖的方法。总的来说，本补助金中提出的项目将通过研究一个强大的调控基因，更详细地了解基本IESC生物学，并推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.