KLF5 regulation of intestinal development and stem cell homeostasis.

描述(由申请人提供)：控制肠道发育和干细胞动态平衡的因素仍然不充分。Kruppel样因子5(KLF5，又称IKLF或BTEB2)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。KLF5在原肠发育过程中的早期胚胎和原始内胚层中高表达，在形态发生和细胞分化过程中在中肠、后肠和胚肠中持续表达。在出生后出现肠道祖细胞(Lieberkuhn的隐窝)期间，KLF5被限制在隐窝内，并且其表达终生维持在这个生殖前体区域。在此背景下，KLF5被认为通过调节细胞周期机制(如细胞周期蛋白)促进增殖，并在致癌转化中发挥作用。然而，KLF5的S在胚胎和成体肠道干细胞自我更新和分化中的作用仍未得到验证。在这里，我们建议检验KLF5控制胚胎和成人肠道干细胞自我更新和分化的假设。在初步研究中，我们建立了转基因小鼠，在其中KLF5可以在肠道中被诱导缺失或激活，作为一种手段来研究KLF5在体内的功能。这些数据表明，KLF5在调节肠道干细胞/祖细胞的增殖和分化方面具有重要作用。我们还建立了一种新的方法来指导人类多能干细胞(PSCs、胚胎干细胞和诱导多能干细胞)在体外发育为肠道器官，并表明KLF5在该系统的关键发育阶段表达。在这项提案中，我们将利用这些独特的资源来确定KLF5在调节肠道自我更新和分化中的作用。目标有三：1)明确KLF5控制肠道发育的必要条件和充分性。假设：KLF5的缺失将导致肠道形态发生和细胞分化受损，而KLF5的表达增加将引导胚胎肠道祖细胞早熟分化。我们将利用人胚胎干细胞来源的肠道器官和转基因小鼠来操纵KLF5的表达，以确定KLF5的S在胚胎肠道发育中的作用。2.)确定KLF5控制成人肠道干细胞动态平衡的必要性和充分性。假设：KLF5的缺失将阻碍干细胞的分化和促进干细胞的扩张，而泛上皮性KLF5将耗尽干细胞库。利用成年转基因小鼠和隐窝来源的上皮器官，将在可诱导的KLF5操作条件下定义肠道干细胞的动态平衡。3.)确定KLF5调节胚胎形态发生和成人肠道稳态的关键靶点。我们已经利用基因芯片和生物信息学方法确定了KLF5的下游效应因子。为了深入了解KLF5的作用机制，将对目标基因进行上位性分析，以测试它们在KLF5可操纵的胚胎和成体肠道器官/类培养中控制分化和内稳态的能力。直接的KLF5靶标将通过DNA结合和启动子报告分析来鉴定。 公共卫生相关性：拟议的研究将确定KLF5是否是未来疗法的靶点，以调节肠道的生长和成熟，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。该项目的结果将扩大对肠道发育和干细胞关键方面的科学知识，并测试一种新的模型，在该模型中，KLF5是肠道上皮形态发生和动态平衡的主要调节者。总而言之，该项目将有助于我们实现调控婴儿和成人肠道生长和成熟的长期目标，并将展示操纵患者来源的PSCs生产用于细胞治疗的肠道组织的可行性。

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.