Genetic Analysis of MicroRNA Functions in Skin Stem Cells In Vivo

项目总结： 哺乳动物的皮肤及其附属物作为身体最外层的屏障来保护 防止体内器官受到环境危害，并使人体内保持必要的液体。 哺乳动物皮肤的动态平衡和完整性由多个祖细胞和干细胞维持 驻留在不同皮肤隔间的细胞群，如毛囊间的基底细胞 毛囊中的表皮和隆起的干细胞。在上皮细胞中，细胞黏附、迁移和 扩散是受许多机制控制的基本属性。在关键中 调节因子，即microRNAs(MiRNAs)是一类发挥重要作用的非编码小RNA 在哺乳动物中，不同细胞类型和组织的基因调控。尽管监管力度不大， 作为单个靶点，miRNAs广泛地调节大量(60%)基因并发挥重要作用 在广泛的生物过程中发挥作用。在哺乳动物的皮肤中，皮肤的关键功能 成体HF胚胎皮肤发育和维持过程中的整个miRNA途径 血统得到了很好的欣赏。相比之下，单个miRNA的知识对其 目标和功能仍然很少。重要的是，与其他监管机构类似，miRNAs如何 调节细胞迁移和增殖还没有在完整皮肤的背景下进行研究 活着的动物。为了解决这些重要问题，我们开发了一些技术来直接 捕获miRNA及其靶向mRNA片段并对细胞迁移和增殖进行成像 以及活体动物完整皮肤中的细胞骨架动力学。使用这些最先进的工具 与我们的小鼠模型一起，我们发现miR-205，在体内表达最高的miRNA 上皮干细胞，通过靶向黏附连接的成分促进细胞迁移， 表皮和毛囊中的肌动蛋白细胞骨架和机械感应基因。在这 项目中，我们将进一步研究miR-205调节的细胞迁移如何改变平衡 表皮增殖和分化之间的关系(目标1)；miR-205如何- 诱导细胞迁移触发幼年和老年小鼠毛囊生长及其增强作用 毛囊生长影响毛囊干细胞(目标2)；并探索Piezo1，a 机械激活的离子通道和一种新的miR-205靶点，控制头发的静止 卵泡干细胞/祖细胞(目标3)。综上所述，这里提出的研究如果成功，将 显著提高我们对由单个miRNAs介导的机制的了解 管理活动物中的细胞迁移和增殖。从这些研究中获得的知识 在正常和紧张的条件下将为操纵miRNA和利用 用于再生医学的上皮干细胞。

Project Summary: Mammalian skin and its appendages function as the outermost barrier of the body to protect inner organs from environmental hazards and keep essential fluids within the body. Homeostasis and integrity of mammalian skin are maintained by multiple progenitor and stem cell populations residing in distinct skin compartments such as basal cells in the interfollicular epidermis and bulge stem cells in hair follicles. In epithelial cells, cell adhesion, migration and proliferation are fundamental properties that are controlled by many mechanisms. Among key regulators, microRNAs (miRNAs) are a class of small, noncoding RNAs that take essential roles in mammalian gene regulation in diverse cell types and tissues. Despite modest regulation of individual targets, miRNAs broadly modulate a large number (60%) of genes and play important roles in a wide range of biological processes. In mammalian skin, the critical functions of the entire miRNA pathway in both embryonic skin development and maintenance of adult HF lineages have been well appreciated. In contrast, the knowledge of individual miRNAs for their targets and function remains scarce. Importantly, similar to other regulators, how miRNAs regulate cell migration and proliferation has not been examined in the context of intact skin in live animals. To address these important issues, we have developed techniques to directly capture miRNA and their targeted mRNA fragments and to image cell migration and proliferation as well as cytoskeleton dynamics in intact skin of live animals. Using these state-of-art tools together with our mouse models, we find that miR-205, the most highly expressed miRNA in epithelial stem cells, promotes cell migration by targeting components of adherens junctions, actin cytoskeleton and mechanosensing genes in both epidermis and hair follicles. In this project, we will further examine how miR-205-regulated cell migration alters the balance between epidermal proliferation and differentiation in the epidermis (Aim 1); how miR-205- induced cell migration triggers hair follicle growth in young and aged mice and how enhanced hair follicle growth affects hair follicle stem cells (Aim 2); and probe how the loss of Piezo1, a mechanically activated ion channel and a new miR-205 target, governs the quiescence of hair follicle stem/progenitor cells (Aim 3). Taken together, studies proposed here, if successful, will significantly enhance our knowledge about mechanisms mediated by individual miRNAs that govern cell migration and proliferation in live animals. The knowledge gained from these studies under normal and stressed conditions will pave the way to manipulate miRNAs and utilize epithelial stem cells for regenerative medicine.