Leveraging Single-Cell Analysis to Elucidate Mechanisms of Vertebrate LimbRegeneration

人类和其他哺乳动物的后天再生能力极其有限，而这些 局限性对健康和生活质量构成了重大挑战。与人类不同的是，紫杉醇火蜥蜴 再生许多器官和附属物，如四肢，取得了惊人的成功。Axolotl的四肢非常 在解剖学上与人类四肢相似，因此它们为发现再生能力提供了一个理想的机会 可能导致未来治疗学发展的机制。在我的新实验室里，我们 研究Axolotls肢体再生的分子机制，以便我们以后应用这一点 理解为什么人类不能再生肢体的知识。 一个悬而未决的问题是，为什么高度再生的有机体使用一种称为胚泡的结构，在这种结构中 体内祖细胞积累，以驱动再生。胚泡细胞在谱系上是异质性的。 很可能是它们的潜力，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胚泡。为了理解这些问题，我们有 启动了一种基于rna-seq的大型方法，我们正在将这种方法与强大的新工具相结合，用于 检测这些生物体中的基因功能。在我们的第一个分析中，我们已经分析了 来自两个关键组织的单个细胞，在一个时间点。我们还生成了一个组织编码的从头开始 转录组作为基因分配和差异基因表达分析的参考。这个 最初的单个细胞测序是完全形成的胚泡细胞和伤口表皮细胞，这过于 胚泡和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，因为此时芽囊线虫种群正处于数量高峰 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文字记录 在这些重要组织中的单个细胞中被特异性上调，我们已经进行了功能 对其中两个基因进行分析。 在这项提案中，我们的目标是使用这一强大的策略来识别 在胚泡形成过程中，支持从完整的组织到激活的祖细胞的过渡。我们会 分析更多单个细胞的转录本，但现在我们将查询从时间点收集的细胞 在截肢和完全胚泡之间。同时，我们将进一步研究第一个发现的基因 分析，特别是那些显示二进制表达模式并因此可以区分 胚泡细胞或伤口表皮细胞。我们将使用最近开发的功能损失和功能增益 在体内，在再生的肢体中询问特定基因的技术。这项工作具有创新性，因为它需要 这是一种发现肢体再生机制的完全先验的方法，它在单细胞上做到了这一点 水平，它利用了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.