Mechanisms driving stem cell responses to injury in planarians

摘要 组织的成功再生需要干细胞可塑性、增殖性和 分化，以产生与先前存在的组织和器官整合的新细胞。路径 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 而再生所需的细胞分化仍然知之甚少。在大多数模式生物中， 再生能力有限，干细胞稀缺，这使得很难准确定位 损伤后干细胞增殖和分化的调控机制。与之形成对比的是，爬行动物 稻纵卷叶蝉有丰富的干细胞，可被损伤和燃料连续激活 再生。像胚胎干细胞一样，脊椎动物干细胞也有能力分化成任何类型的 组织。这些多能干细胞可以很容易地识别、监测、纯化和彻底剖析 分子水平。我们最近有了两项重要发现，它们构成了这一提议的基础。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，因为它阻止了细胞周期，而且减少了 干细胞会发生凋亡。其次，我们开创了一种化学方法，选择性地移除单个器官， 咽部。咽再生需要保守的Forkhead转录因子上调 在这种靶向损伤后立即在干细胞的离散子集中注射FOXA。我们发现细胞外的 信号调节激酶(ERK)是这些行为的中心驱动因素。ERK促进培养细胞分化 干细胞，但它是如何在受伤后被激活的，人们知之甚少。总之，这些发现确立了我们的 中心假说，即损伤使细胞周期同步，使局部信号能够引导干细胞 分化成离散的细胞命运。在目标1中，我们将确定损伤如何诱导干细胞细胞周期停滞。 辐射后的细胞。我们将检查DNA修复，并测试上调的保守基因的功能 受伤后。在目标2中，我们将剖析通过纯化和促进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。我们将识别富含在这些受体上的 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，我们将 识别上游受体，通过RNAi的组合激活干细胞中的MAP激酶信号， 药理学和生物化学。这项提议利用了我们用精确的侮辱来挑战干细胞的能力， 提供了一个镜头，了解在损伤和动态平衡期间实现灵活干细胞反应的机制。 了解在生理相关背景下支配干细胞行为的分子机制 将为未来再生医学技术战略的设计提供信息。

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.