Leveraging Single-Cell Analysis to Elucidate Mechanisms of Vertebrate LimbRegeneration

人类和其他哺乳动物的产后再生能力极其有限， 限制对健康和生活质量构成重大挑战。与人类相比，蝾螈 再生许多器官和附肢，如四肢，取得了惊人的成功。蝾螈的四肢 在解剖学上类似于人类的四肢，因此它们提供了一个理想的机会， 可能导致未来治疗方法发展的机制。在我的新实验室里， 研究蝾螈肢体再生的分子机制， 了解为什么人类不能再生肢体。 一个悬而未决的问题是，为什么高度再生的生物体使用一种称为芽基的结构， 内部祖细胞聚集，以驱动再生。芽基细胞的谱系是异质的 以及它们的潜力，但人们对这些属性是如何控制的，甚至是如何控制的知之甚少。 祖细胞被提示激活并加入芽基。为了理解这些问题，我们有 我们启动了一种基于大型RNA-seq的方法，我们正在将这种方法与强大的新工具结合起来， 检测这些生物体的基因功能。在我们的第一个分析中，我们已经分析了 两个关键组织的单个细胞。我们还生成了一个组织编码的 转录组用作基因分配和差异基因表达分析的参考。的 测序的初始单个细胞是完全形成的芽基细胞和伤口表皮细胞，它们覆盖在表皮细胞上。 芽基，被认为控制再生的关键方面。我们选择了这个时间点，23天后- 截肢，作为第一个采样点，因为在这个时候，芽基人口是在其高度的数字 但还没有任何明显的分化迹象。到目前为止，我们已经发现了许多 在这些重要组织中的单个细胞中特异性上调，我们已经进行了功能性研究， 分析了其中两个基因。 在这项提议中，我们的目标是使用这种强大的策略来识别基因表达的变化， 支持在胚基产生期间从完整组织向活化祖细胞的转变。我们将 分析更多单个细胞的转录组，但现在我们将查询从时间点收集的细胞， 截肢和完整芽基之间的关系与此同时，我们将进一步研究在第一个实验中发现的基因。 分析，特别是那些显示二进制表达模式，因此可以区分亚型， 芽基细胞或伤口表皮细胞。我们将使用最近开发的功能丧失和功能获得 在再生肢体的体内询问特定基因的技术。这项工作是创新的，因为它需要 这是一种完全先验的方法来发现肢体再生的机制，它是在单细胞上实现的 水平，它利用强大的新技术来检查基因功能。

Humans and other mammals have extremely limited postnatal regenerative abilities, and these limitations pose a significant challenge to health and quality of life. In contrast to humans, axolotl salamanders regenerate many organs and appendages, such as limbs, with astonishing success. Axolotl limbs are very similar anatomically to human limbs, so they offer an ideal opportunity for discovering regenerative mechanisms that might lead to the development of future therapeutics. In my new laboratory, we are investigating the molecular mechanisms of limb regeneration in axolotls so that we can later apply this knowledge to understanding why humans cannot regenerate limbs. An outstanding question is why highly-regenerative organisms use a structure called a blastema, where internal progenitor cells accumulate, to drive regeneration. Blastema cells are heterogeneous in their lineage and likely their potentials, but very little is known about how these attributes are controlled or even how progenitor cells are cued to become activated and join the blastema. To understand these questions, we have initiated a large RNA-seq based approach, and we are coupling this approach to powerful new tools for examining gene function in these organisms. In our first analysis, we have profiled the transcriptomes of individual cells from two key tissues, at one time point. We also generated a tissue-coded de novo transcriptome to use as a reference for gene assignment and for differential gene expression analysis. The initial individual cells sequenced were fully-formed blastema cells and wound epidermis cells, which overly the blastema and are thought to control key aspects of regeneration. We chose this time point, 23 days post- amputation, as the first sampling point because at this time the blastema population is at its height for numbers of cells but there are not yet any overt signs of differentiation. We have thus far discovered many transcripts that are specifically upregulated in individual cells in these important tissues, and we have performed functional analyses with two of the genes. In this proposal, we aim to use this powerful strategy to identify the gene expression changes that support the transition from intact tissue to activated progenitor cells during the creation of the blastema. We will profile the transcriptomes of more individual cells, but now we will query cells harvested from time points between amputation and the full blastema. In parallel, we will further examine genes uncovered in the first analysis, specifically those that show binary expression patterns and may therefore distinguish subtypes of blastema cells or wound epidermis cells. We will use recently-developed loss-of-function and gain-of-function technolgies to interrogate specific genes in vivo, in regenerating limbs. This work is innovative because it takes a completely a priori approach to discovering mechanisms of limb regeneration, it does so at the single-cell level, and it capitalizes on powerful new techniques for examining gene function.