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 再生所必需的细胞增殖和分化仍然知之甚少。在大多数 模型生物，再生能力有限，干细胞稀缺，这使得很难 明确损伤后调节干细胞增殖和分化的机制。相比之下， 扁形虫Schmidteamidiomedioea有丰富的干细胞，这些干细胞被损伤和燃料激活 连续再生与胚胎干细胞一样， into any type类型oftissue组织.这些多能干细胞可以容易地被鉴定、监测、纯化，并 彻底剖析了分子水平。我们最近做出了两项重要发现，构成了 这一提议的基础。首先，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， 因为它停止了细胞周期，较少的干细胞发生凋亡。第二，我们开创了一种化学物质 选择性切除咽部这一单一器官的方法。咽部再生需要上调 保守的Forkhead转录因子FoxA在一个离散的干细胞亚群中， 这种针对性的伤害。我们发现细胞外信号调节激酶（ERK）是这些信号转导的中心驱动因子。 行为。ERK在培养的干细胞中促进分化，但在损伤后如何激活还不清楚。 明白总之，这些发现确立了我们的中心假设，即损伤是由神经系统引起的。 细胞周期，使当地的线索渠道干细胞分化向离散细胞的命运。在目标1中， 我们将确定辐射后损伤如何诱导干细胞的细胞周期停滞。我们会检查DNA 修复和测试损伤后上调的保守基因的功能。在目标2中，我们将剖析 通过茎的纯化和单细胞测序驱动器官特异性再生的机制 器官丧失后细胞增殖我们将鉴定这些细胞上富集的受体，并测试它们的功能。 以确定它们是否作用于FoxA的上游。在目标3中，我们将确定上游 在干细胞中激活MAP激酶信号传导的受体与RNAi、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， 透镜的机制，使灵活的干细胞反应损伤和稳态。 了解在生理学相关的环境中控制干细胞行为的分子机制 背景将为再生医学技术未来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 proliferationand 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 oftissue. These pluripotent stem cells can be readily identified, monitored, purified, and thoroughly profiled at themolecular 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 stemcells after radiation. We will examine DNA repair and test the function of conserved genes that are upregulatedafter 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.