KLF5 regulation of intestinal development and stem cell homeostasis

KLF5 对肠道发育和干细胞稳态的调节

• 批准号: 8905197
• 负责人: NOAH Freeman SHROYER
• 金额: $ 40.22万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 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 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8905197
• 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 homeostasis; 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.

描述（由申请人提供）：控制肠道发育和干细胞稳态的因素仍然没有充分表征。Kruppel样因子5（KLF 5，也称为IKLF或BTEB 2）是一种锌指转录因子，被认为调节胃肠道上皮细胞的增殖和分化。KLF 5在早期胚胎和原肠胚形成期间的原始内胚层中高度表达，在形态发生和细胞分化期间在中/后肠和胚胎肠中持续表达。在出生后肠祖细胞区（Lieberkuhn隐窝）出现期间，KLF 5被限制在隐窝中，并且其表达在整个生命中维持在该祖细胞区中。在这种情况下，KLF 5被假设为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，KLF 5在胚胎和成人肠干细胞自我更新和分化中的作用尚未得到证实。在这里，我们建议测试的假设，KLF 5控制自我更新和分化的胚胎和成人肠干细胞。在初步研究中，我们开发了转基因小鼠，其中KLF 5可以在肠道中诱导缺失或激活，作为研究KLF 5体内功能的手段。这些数据表明KLF 5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的方法来指导人类多能干细胞（PSC，胚胎干细胞和诱导多能干细胞）在体外发育成肠类器官，并表明KLF 5在该系统的关键发育步骤中表达。在这项提案中，我们将利用这些独特的资源来确定KLF 5在调节肠道自我更新和分化中的作用。三个目标是：（1）。明确KLF 5控制肠道发育的要求和充分性。假设：KLF 5的缺失将导致肠形态发生和细胞分化受损，而KLF 5表达增加将指导胚胎肠祖细胞早熟分化。我们将操纵KLF 5的表达，以确定KLF 5在胚胎肠道发育中的作用，使用人PSC衍生的肠道类器官和转基因小鼠。2.）的情况。确定KLF 5控制成体肠干细胞稳态的需求和充分性。假设：KLF 5的缺失将阻断干细胞的分化并促进干细胞的扩增，而泛上皮KLF 5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，将在诱导型KLF 5操纵条件下定义肠干细胞稳态。3.）第三章确定KLF 5调节胚胎形态发生和成人肠道稳态的关键靶点。我们已经确定了假定的下游效应KLF 5使用微阵列和生物信息学的方法。为了深入了解KLF 5的作用机制，将对靶基因进行上位性分析，以测试它们在KLF 5可操作的胚胎和成人肠道器官/类细胞培养物中控制分化和稳态的能力。直接KLF 5靶标将通过DNA结合和启动子报告基因试验进行鉴定。

项目成果

Kruppel-like factor 5 controls villus formation and initiation of cytodifferentiation in the embryonic intestinal epithelium.

• DOI: 10.1016/j.ydbio.2012.12.010
• 发表时间: 2013-03-15
• 期刊: DEVELOPMENTAL BIOLOGY
• 影响因子: 2.7
• 作者: Bell, Sheila M.;Zhang, Liqian;Xu, Yan;Besnard, Valerie;Wert, Susan E.;Shroyer, Noah;Whitsett, Jeffrey A.
• 通讯作者: Whitsett, Jeffrey A.

Respiratory epithelial cells orchestrate pulmonary innate immunity.

• DOI: 10.1038/ni.3045
• 发表时间: 2015-01
• 期刊: Nature immunology
• 影响因子: 30.5