KLF5 regulation of intestinal development and stem cell homeostasis.

KLF5 调节肠道发育和干细胞稳态。

• 批准号: 8162496
• 负责人: NOAH Freeman SHROYER
• 金额: $ 44.94万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-05 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8162496
• 关键词: Adult; Bioinformatics; Biological Assay; Brush Border; Cell Cycle; Cell Therapy; Cells; Cyclins; DNA Binding; Data; Development; ELF3 gene; Embryo; Endoderm; Enterocytes; Enteroendocrine Cell; Epithelial; Failure; Future; Gene Targeting; Genes; Genetic Epistasis; Goals; Goblet Cells; Growth; Hindgut; Homeostasis; Human; Human Development; In Vitro; Infant; Intestinal Diseases; Intestines; Knowledge; Life; Methods; Modeling; Morphogenesis; Mus; Necrotizing Enterocolitis; Oncogenic; Organ; Organoids; Paneth Cells; Patients; Pluripotent Stem Cells; Regulation; Reporter; Resources; Role; Stem Cell Development; Stem cells; Structure of intestinal gland; System; Testing; Tissues; Transgenic Mice; Villus; Zinc Fingers; embryonic stem cell; gastrointestinal epithelium; gastrulation; in vivo; induced pluripotent stem cell; insight; intestinal villi; novel; postnatal; prevent; progenitor; promoter; research study; self-renewal; stem cell division; transcription factor

DESCRIPTION (provided by applicant): The factors that control intestinal development and stem cell homeostasis remain inadequately characterized. Kruppel like factor 5 (KLF5, also termed IKLF or BTEB2) is a zinc-finger transcription factor that is thought to regulate proliferation and differentiation of gastrointestinal epithelia. KLF5 is highly expressed in the early embryo and the primordial endoderm during gastrulation, with continued expression in the mid/hindgut and embryonic intestine during morphogenesis and cytodifferentiation. During postnatal emergence of intestinal progenitor do- mains (crypts of Lieberkuhn), KLF5 becomes restricted to the crypts and its expression is maintained in this pro- genitor zone throughout life. In this context, KLF5 is hypothesized to promote proliferation by regulating cell cycle machinery such as cyclins, and has a role in oncogenic transformation. However, KLF5's role in embryonic and adult intestinal stem cell self-renewal and differentiation remains untested. Here, we propose to test the hypothesis that KLF5 controls self-renewal and differentiation of both embryonic and adult intestinal stem cells. In preliminary studies, we developed transgenic mice in which KLF5 can be inducibly deleted or activated in the intestine, as a means to investigate the function of KLF5 in vivo. These data show that KLF5 has an essential role in regulating proliferation and differentiation of intestinal stem cell/progenitors. We have also established a novel method to direct development of human pluripotent stem cells (PSCs, embryonic stem cells and induced pluripotent stem cells) into intestinal organoids in vitro, and show that KLF5 is expressed at key developmental steps in this system. In this proposal, we will utilize these unique resources to define the role of KLF5 in regulating self-renewal and differentiation in the intestine. Three aims will be pursued: 1.) Define the requirement and sufficiency for KLF5 to control intestinal development. Hypothesis: Loss of KLF5 will result in impaired intestinal morphogenesis and cytodifferentiation, whereas increased expression of KLF5 will direct embryonic intestinal progenitors to precociously differentiate. We will manipulate KLF5 expression in order to define KLF5's role in embryonic intestinal development, using human PSC-derived intestinal organoids and transgenic mice. 2.) Determine the requirement and sufficiency for KLF5 to control adult intestinal stem cell homeostasis. Hypothesis: Loss of KLF5 will block differentiation and promote expansion of stem cells, whereas pan-epithelial KLF5 will deplete the stem cell pool. Using adult transgenic mice and crypt-derived epithelial organoids, intestinal stem cell homeostasis will be defined under conditions of inducible KLF5 manipulation. 3.) Define key targets of KLF5 that regulate embryonic morphogenesis and adult intestinal homeostasis. We have identified putative downstream effectors of KLF5 using microarray and bioinformatic approaches. To gain insight into the mechanisms of KLF5 action, epistasis analysis will be performed with target genes to test their ability to control differentiation and homeostasis in KLF5-manipulatable embryonic and adult intestinal organ/oid culture. Direct KLF5 targets will be identified by DNA binding and promoter reporter assays. PUBLIC HEALTH RELEVANCE: The proposed studies will determine whether KLF5 is a target for future therapies to regulate growth and maturation of the intestine as a means to prevent and treat intestinal diseases such as necrotizing enterocolitis and intestinal failure. The results of this project will expand scientific knowledge of key aspects of intestinal development and stem cells, and test a new model in which KLF5 is a master regulator of intestinal epithelial morphogenesis and homeostasis. Together, this project will contribute to our long-term goals to regulate growth and maturation of the intestine in infants and adults, and will demonstrate the feasibility of manipulating patient-derived PSCs to produce intestinal tissues for cell therapy.

描述（由申请人提供）：控制肠道发育和干细胞稳态的因素仍未得到充分表征。 Kruppel 样因子 5（KLF5，也称为 IKLF 或 BTEB2）是一种锌指转录因子，被认为可以调节胃肠道上皮细胞的增殖和分化。 KLF5 在原肠胚形成过程中在早期胚胎和原始内胚层中高度表达，并在形态发生和细胞分化过程中在中/后肠和胚胎肠中持续表达。在出生后肠道祖细胞区域（Lieberkuhn 隐窝）出现期间，KLF5 被限制在隐窝中，并且其表达在整个生命过程中都维持在该祖细胞区域。在此背景下，假设 KLF5 通过调节细胞周期机制（如细胞周期蛋白）来促进增殖，并在致癌转化中发挥作用。然而，KLF5 在胚胎和成体肠道干细胞自我更新和分化中的作用尚未得到测试。在这里，我们建议检验 KLF5 控制胚胎和成体肠道干细胞的自我更新和分化的假设。在初步研究中，我们开发了转基因小鼠，其中KLF5可以在肠道中诱导删除或激活，作为研究KLF5体内功能的一种手段。这些数据表明KLF5在调节肠干细胞/祖细胞的增殖和分化中具有重要作用。我们还建立了一种新方法，可在体外指导人类多能干细胞（PSC、胚胎干细胞和诱导多能干细胞）发育成肠道类器官，并表明 KLF5 在该系统的关键发育步骤中表达。在本提案中，我们将利用这些独特的资源来定义 KLF5 在调节肠道自我更新和分化中的作用。我们将追求三个目标：1.) 明确 KLF5 控制肠道发育的要求和充分性。假设：KLF5 的缺失将导致肠道形态发生和细胞分化受损，而 KLF5 表达增加将引导胚胎肠道祖细胞早熟分化。我们将使用人类 PSC 衍生的肠道类器官和转基因小鼠来操纵 KLF5 表达，以确定 KLF5 在胚胎肠道发育中的作用。 2.) 确定 KLF5 控制成体肠道干细胞稳态的需求和充分性。假设：KLF5 的缺失将阻碍干细胞的分化并促进干细胞的扩增，而泛上皮 KLF5 将耗尽干细胞库。使用成年转基因小鼠和隐窝衍生的上皮类器官，将在可诱导的 KLF5 操作条件下定义肠干细胞稳态。 3.) 定义 KLF5 调节胚胎形态发生和成人肠道稳态的关键靶点。我们使用微阵列和生物信息学方法鉴定了 KLF5 的假定下游效应子。为了深入了解 KLF5 的作用机制，将对目标基因进行上位分析，以测试它们在 KLF5 可操纵的胚胎和成人肠道器官/类培养物中控制分化和稳态的能力。直接 KLF5 靶点将通过 DNA 结合和启动子报告基因检测来鉴定。 公共健康相关性：拟议的研究将确定 KLF5 是否是未来调节肠道生长和成熟疗法的靶标，作为预防和治疗坏死性小肠结肠炎和肠衰竭等肠道疾病的手段。该项目的结果将扩展肠​​道发育和干细胞关键方面的科学知识，并测试一种新模型，其中 KLF5 是肠上皮形态发生和稳态的主要调节因子。总之，该项目将为我们调节婴儿和成人肠道生长和成熟的长期目标做出贡献，并将证明操纵患者来源的 PSC 来生产用于细胞治疗的肠道组织的可行性。