Mechanisms driving stem cell responses to injury in planarians

涡虫干细胞对损伤反应的驱动机制

• 批准号: 10264039
• 负责人: Carolyn Elizabeth Adler
• 金额: $ 37.17万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-15 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10264039
• 关键词: Amputation; Animal Model; Animals; Apoptosis; Automobile Driving; Behavior; Biochemistry; Biological Assay; Cell Cycle; Cell Cycle Arrest; Cell Differentiation process; Cell division; Cells; Chemicals; Cues; DNA; DNA Repair; Epidermal Growth Factor Receptor; Extracellular Signal Regulated Kinases; Fibroblast Growth Factor Receptors; Foundations; Future; G2/M Checkpoint Pathway; Genes; Goals; Homeostasis; Injury; Kinetics; Ligands; Methods; Microtubule Depolymerization; Mitogen-Activated Protein Kinases; Molecular; Monitor; Myocardial Infarction; Natural regeneration; Nocodazole; Organ; Pathway interactions; Pharmacology; Pharyngeal structure; Phosphorylation; Physiological; Planarians; Platyhelminths; Pluripotent Stem Cells; Proliferating; RNA Interference; RNA interference screen; Radiation; Radiation Injuries; Regenerative Medicine; Regenerative capacity; Regulator Genes; Signal Pathway; Signal Transduction; Stroke; Surgical Injuries; Technology; Testing; Tissues; Transcript; Transcriptional Activation; Up-Regulation; cell behavior; design; embryonic stem cell; flexibility; forkhead protein; improved; injured; knock-down; lens; organ regeneration; progenitor; receptor; regenerative approach; response; response to injury; single cell sequencing; single-cell RNA sequencing; stem cell differentiation; stem cell population; stem cell proliferation; stem cells; tissue regeneration; transcriptome; transcriptome sequencing

Abstract Successful regeneration of tissues requires transient increases in stem cell plasticity, proliferation, and differentiation, in order to produce new cells that integrate with preexisting tissues and organs. Pathways governing these critical behaviors have been identified, but how injury signals can trigger stem cell proliferation and differentiation of cells necessary for regeneration remains poorly understood. In most model organisms, regenerative capacity is limited and stem cells are scarce, which has made it difficult to pinpoint the mechanisms regulating stem cell proliferation and differentiation after injury. By contrast, the planarian flatworm Schmidtea mediterranea has abundant stem cells that are activated by injury and fuel continuous regeneration. Like embryonic stem cells, planarian stem cells have the capacity to differentiate into any type of tissue. These pluripotent stem cells can be readily identified, monitored, purified, and thoroughly profiled at the molecular level. We recently made two important discoveries that form the foundation of this proposal. First, injury of any type appears to protect stem cells from lethal radiation, because it halts the cell cycle and fewer stem cells undergo apoptosis. Second, we pioneered a chemical method to selectively remove a single organ, the pharynx. Pharynx regeneration requires the upregulation of the conserved Forkhead transcription factor FoxA in a discrete subset of stem cells immediately after this targeted injury. We find that the extracellular signal-regulated kinase (ERK) is a central driver of these behaviors. ERK promotes differentiation in cultured stem cells, but how it is activated after injury is poorly understood. Together, these findings establish our central hypothesis, which is that injury synchronizes the cell cycle, enabling local cues to channel stem cell differentiation toward discrete cell fates. In Aim 1, we will determine how injury induces cell cycle arrest in stem cells after radiation. We will examine DNA repair and test the function of conserved genes that are upregulated after injury. In Aim 2, we will dissect the mechanisms driving organ-specific regeneration by purification and single-cell sequencing of stem cells proliferating after organ loss. We will identify receptors enriched on these cells, and test their function in organ regeneration to determine if they act upstream of FoxA. In Aim 3, we will identify the upstream receptors that activate MAP kinase signaling in stem cells with combinations of RNAi, pharmacology and biochemistry. This proposal exploits our ability to challenge stem cells with precise insults, providing a lens into the mechanisms that enable flexible stem cell responses during injury and homeostasis. Understanding the molecular mechanisms that govern stem cell behavior in a physiologically-relevant context will inform the design of future strategies for regenerative medicine technologies.

摘要 组织的成功再生需要干细胞可塑性、增殖和分化的瞬时增加。 分化，以便产生与先前存在的组织和器官整合的新细胞。途径 控制这些关键行为的机制已经确定，但损伤信号如何触发干细胞增殖 并且对于再生所必需的细胞的分化仍然知之甚少。在大多数模式生物中， 再生能力有限，干细胞稀缺，这使得很难确定 损伤后调节干细胞增殖和分化的机制。相比之下， 扁虫Schmidtea mediterranea具有丰富的干细胞，这些干细胞被损伤激活并持续提供燃料。 再生与胚胎干细胞一样，真涡虫干细胞具有分化成任何类型的细胞的能力。 组织.这些多能干细胞可以容易地鉴定、监测、纯化，并在体外进行彻底的分析。 分子水平。我们最近有两个重要的发现，构成了这个提议的基础。第一、 任何类型的损伤似乎都能保护干细胞免受致命辐射的伤害，因为它会停止细胞周期， 干细胞经历凋亡。其次，我们开创了一种化学方法，选择性地切除一个器官， 咽部咽再生需要上调保守的Forkhead转录因子 在这种靶向损伤后立即在干细胞的离散亚群中表达FoxA。我们发现细胞外 信号调节激酶（ERK）是这些行为的中心驱动力。ERK促进细胞分化 干细胞，但它是如何激活损伤后知之甚少。总之，这些发现确立了我们的 中心假设，即损伤扰乱细胞周期，使局部线索能够引导干细胞 向离散细胞命运分化。在目标1中，我们将确定损伤如何诱导干细胞中的细胞周期阻滞， 辐射后的细胞我们将研究DNA修复和测试保守基因的功能， 伤后在目标2中，我们将剖析通过纯化驱动器官特异性再生的机制， 器官丧失后干细胞增殖的单细胞测序。我们将鉴定出这些受体 细胞，并测试它们在器官再生中的功能，以确定它们是否作用于FoxA的上游。在目标3中，我们 用RNAi的组合鉴定在干细胞中激活MAP激酶信号传导的上游受体， 药理学和生物化学。这项提议利用了我们用精确的损伤来挑战干细胞的能力， 提供了一个透镜进入机制，使灵活的干细胞反应在损伤和稳态。 了解在生理相关背景下支配干细胞行为的分子机制 将为再生医学技术的未来战略设计提供信息。