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）：控制肠发展和干细胞稳态的因素仍然不足。 Kruppel像因子5（KLF5，也称为IKLF或BTEB2）是一个锌指转录因子，被认为可以调节胃肠道上皮的增殖和分化。 KLF5在早期胚胎和原始内胚层中高度表达，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。在产后出现的肠道祖细胞（Lieberkuhn的隐窝）的出现中，KLF5限制在隐窝中，其表达在整个生命周期中都保持在此生长区域。在这种情况下，假设KLF5通过调节细胞周期机制（例如细胞周期蛋白）来促进增殖，并在致癌转化中起作用。但是，KLF5在胚胎和成人肠干细胞自我更新和分化中的作用尚未经过测试。在这里，我们建议检验KLF5控制胚胎和成年肠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，其中可以在肠道中删除或激活KLF5，以作为研究体内KLF5的功能的一种手段。这些数据表明，KLF5在调节肠道干细胞/祖细胞的增殖和分化中具有至关重要的作用。我们还建立了一种新的方法，可以将人多能干细胞（PSC，胚胎干细胞和诱导多能干细胞）在体外进行肠道器官，并表明KLF5在该系统的关键发展步骤中表达。在此提案中，我们将利用这些独特的资源来定义KLF5在调节肠中自我更新和差异化中的作用。将追求三个目标：1。）定义KLF5控制肠道发育的要求和足够的需求。假设：KLF5的丧失将导致肠形形态发生和细胞分化受损，而KLF5的表达增加将导致胚胎肠道祖细胞早熟地分化。我们将使用人类PSC衍生的肠道类器官和转基因小鼠来定义KLF5的表达，以定义KLF5在胚胎肠发育中的作用。 2.）确定KLF5控制成年肠干细胞体内平衡的需求和足够的需求。假设：KLF5的丧失将阻止分化并促进干细胞的扩张，而泛上皮KLF5会耗尽干细胞池。使用成年转基因小鼠和隐窝衍生的上皮器官，将在诱导型KLF5操纵的条件下定义肠道干细胞稳态。 3.）定义调节胚胎形态发生和成年肠内稳态的KLF5的关键靶标。我们已经使用微阵列和生物信息学方法确定了KLF5的推定下游效应子。为了深入了解KLF5作用的机制，将使用靶基因进行上位性分析，以测试其控制KLF5操纵性胚胎和成人肠道和成人肠道器官/OID培养的分化和稳态的能力。 DNA结合和启动子记者测定法将确定直接的KLF5靶标。 公共卫生相关性：拟议的研究将确定KLF5是否是未来疗法调节肠道生长和成熟的靶标，作为预防和治疗肠道疾病（例如坏死性小肠结肠炎和肠道衰竭）的手段。该项目的结果将扩大有关肠道发育和干细胞关键方面的科学知识，并测试一个新模型，其中KLF5是肠上皮形态发生和稳态的主要调节剂。该项目将共同有助于我们的长期目标，以调节婴儿和成人肠的生长和成熟，并将证明操纵患者衍生的PSC以生产肠道组织以进行细胞治疗的可行性。