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稳态的因素仍然没有充分表征。Kruppel样因子5 （KLF5，也称为IKLF或BTEB2）是一种锌指转录因子，被认为调节胃肠道上皮细胞的增殖和分化。KLF5在早期胚胎和原肠胚形成期间高表达，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（Lieberkuhn隐窝）的出现过程中，KLF5被限制在隐窝中，其表达终生维持在这个祖细胞区。在这种情况下，KLF5被假设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验证。在这里，我们提出验证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KLF5在肠道中被诱导删除或激活的转基因小鼠，作为研究KLF5在体内功能的手段。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）在体外发育为肠道类器官，并表明KLF5在该系统的关键发育阶段表达。在本提案中，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。将追求三个目标：1)确定KLF5控制肠道发育的需要量和充分性。假设：KLF5缺失会导致肠道形态发生和细胞分化受损，而KLF5表达增加会导致胚胎肠道祖细胞过早分化。我们将利用人类psc衍生的肠道类器官和转基因小鼠来操纵KLF5的表达，以确定KLF5在胚胎肠道发育中的作用。2)。确定KLF5在控制成体肠道干细胞稳态中的需要量和充分性。假设：KLF5的缺失会阻碍干细胞的分化并促进干细胞的扩增，而泛上皮KLF5则会耗尽干细胞库。利用成年转基因小鼠和隐窝来源的上皮类器官，将在可诱导的KLF5操纵条件下确定肠道干细胞的稳态。3)。确定KLF5调控胚胎形态发生和成人肠道稳态的关键靶点。我们已经使用微阵列和生物信息学方法确定了KLF5的推定下游效应物。为了深入了解KLF5的作用机制，将对靶基因进行上位性分析，以测试它们在KLF5可操纵的胚胎和成人肠道器官/样培养中控制分化和稳态的能力。直接的KLF5靶点将通过DNA结合和启动子报告子检测来鉴定。

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.