Genetic Control of Intestinal Epithelial Stem Cell Maintenance & Differentiation

描述（由申请人提供）：肠上皮由位于隐窝基部的多能肠上皮干细胞（IESCs）池不断更新。IESCs的定义是：a)自我更新的能力，以及b)产生所有终末分化肠上皮谱系的多能性，这些特征也被称为“干性”。IESC生物学中的一个主要问题是是什么控制了干性。Sox （Sry Box）转录因子家族的成员在其他组织中作为主干控制开关发挥作用。本提案旨在明确Sox9在IESC干性中的具体作用和基因靶点。我们最近证明了不同水平的Sox9差异标记功能IESCs、祖细胞、肠内分泌细胞和Paneth细胞。Sox9和Lgr5都标记了嵌入Paneth细胞之间并与Paneth细胞密切接触的IESCs。新的证据表明，Paneth细胞充当“护理”细胞，对IESC的维持至关重要。胚胎发生期间肠上皮中Sox9的消融导致Paneth细胞的丢失和小肠和结肠隐窝内增殖增加。我们关于成人肠上皮诱导Sox9消融短期效应的新初步数据证实了隐窝过度增生，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、Paneth细胞中杯状生物标志物Muc2的错误表达、隐窝型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。正常小肠与诱导条件Sox9突变体小肠的基因芯片分析表明，Sox9控制着参与增殖、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切除了Sox9，因此他们没有区分IESC中Sox9的缺失与其他表达Sox9的细胞系（包括Paneth细胞）的表型效应。核心假设是，Sox9通常限制IESC增殖并保持多能性，通过a) IESC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生态位来调节Paneth细胞特异性基因，这些基因在外部控制IESC增殖或效力。为了验证这一假设，我们提出以下目标：1)确定Sox9在IESCs中的具体作用；2)确定Sox9调节Paneth细胞中的基因如何影响IESC的干细胞性；3)确定Sox9在a) IESCs和b) Paneth细胞中调节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。这些发现可能会扩展到与其他组织特异性干细胞相关的平行研究领域。

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.