A single-cell multiomic approach in planarians to understand regeneration.

许多动物在受伤时能够再生缺失的身体部位和器官。这种能力在人类和其他哺乳动物中大大降低，在淡水浮游生物等动物中也存在。甲虫可以在几天内再生出身体的任何部位。由于其实验的适应性和研究的易用性，稻草已经成为研究再生的一个强大的实验室模型。了解这一过程是再生医学的基础，因为它试图在实验室和最终在临床上用人类细胞重建它。平面动物的再生要归功于成年阶段存在的干细胞群体，即所谓的新生母细胞。多年来的研究揭示了基因、信号、分子和过程对脊椎动物的再生非常重要。然而，这些研究缺乏细胞分辨率：我们不知道这些信号在哪些细胞类型中活跃，以及不同类型的细胞在再生过程中如何协调，形成像头部或尾巴一样复杂的组织。这在一定程度上是因为目前从基因上剖析这一过程的技术通常会将所有组织和细胞类型混合到大样本中。其他具有高细胞分辨率的技术存在吞吐量低的问题，在每次分析中只提供几个基因的信息。我们最近帮助开发并使用了一套非常新颖的技术，这套技术正在给生物学带来革命性的变化：单细胞分析。它允许通过对信使RNA和每个细胞的基因调控元件进行测序来分析数千个单个细胞的遗传信息。有了这一点，我们可以对主要的脊椎动物细胞类型进行分类，如肌肉、神经元、表皮和其他几十种，并重建它们的发育过程。这些方法也被用于其他发育系统，如青蛙、鱼和蝾螈，引领了干细胞和发育生物学的单细胞革命。我们已经建立的这种方法的最新版本允许我们在每个实验中分析大约10-100倍的细胞，并使进行重复实验的大样本集成为可能。这是获得有统计学意义的信息的关键修改。因此，为了了解参与这一过程的单个细胞水平上的脊椎动物再生是如何工作的，我们将从不同的再生时间、身体部位和复制中获得大约50万个细胞的单细胞分析图谱。分析所有这些信息将使我们能够阐明每种细胞类型中哪些基因被激活，以及在这个过程中的什么时间点。我们将使用成熟的浮游生物方法来扰乱这些基因的功能，以从扰动的影响中获得关于这些基因作用的功能信息。我们将通过进一步的单细胞转录实验来分析这些基因。所有这些实验都将告诉我们每种细胞类型在再生过程中的表现。有了这样的技术和细胞数量，即使是最稀有的细胞类型也将被捕获。我们将阐明它们用来指示再生细胞整合到新的再生组织中的信号，以及这些信号的功能影响。总之，这些实验将使我们能够以前所未有的定量方式，在单细胞水平上，利用功能信息来分析脊椎动物的再生。这将是了解动物再生的重要一步。我们的数据将刺激对浮游生物的新研究，研究我们发现的调控因素和过程，并在其他动物的动物再生研究中引领方向。我们将首次使用的工具和方法将成为再生和发育过程研究的标准，也是单细胞生物学的关键。了解动物是如何再生的是推进人类再生医学议程的关键。

Many animals are able to regenerate missing body parts and organs when they are injured. This ability, very reduced in humans and other mammals, is present in animals such as freshwater planarians. The planarian Schmidtea mediterranea can regenerate any body part in a matter of days. Thanks to its amenability for experimentation and ease of research use, S. mediterranea has become a powerful laboratory model where to study regeneration. Understanding this process is fundamental for regenerative medicine, as it seeks to recreate it with human cells in the laboratory, and ultimately in the clinic.Planarians regenerate thanks to a stem cell population present in their adult stages, the so-called neoblasts. Years of research have revealed genes, signals, molecules and processes that are important for planarian regeneration. However, these studies have suffered from the lack of cellular resolution: we do not know in which cell types these signals are active and how the different cell types coordinate during the regeneration process to form a tissue as complex as a head or a tail. Partly this is because current techniques to dissect the process genetically typically blend all tissues and cell types into bulk samples. Other techniques with high cellular resolution suffer from low throughput, giving information of a few genes in each assay.We have recently helped develop and used a very novel set of techniques that are revolutionising biology: single-cell analysis. It allows profiling the genetic information of thousands of individual cells by sequencing messenger RNAs and gene regulatory elements from each of them. With this, we can classify the major planarian cell types such as muscle, neurons, epidermis and dozens more and reconstruct their developmental processes. These methods have also been used in other developmental systems such as frogs, fish and newts, leading the way to a single-cell revolution of stem cell and developmental biology. The latest version of this method that we have already set up allow us to profile ~10-100 times more cells per experiment and makes feasible performing large sample sets with replicate experiments. This is a key modification to obtain statistically significant information.Thus, to understand how planarian regeneration works at the level of the individual cells that participate in the process, we will obtain single cell analysis profiles of around half a million cells, from different regeneration times, body parts and in replicates. Analysing all this information will allow us to elucidate which genes are activated in each cell type and at what time points during the process. We will perturb the function of these genes using well-established planarian methods to obtain functional information about the role of these genes from the effects of the perturbation. We will analyse these by further single-cell transcriptomic experiments. All of these experiments will tell us how each individual cell type behaves in the regeneration process. With such technique and cell numbers, even the rarest cell types will be captured. We will elucidate the signals they use to instruct the regenerative cells to integrate in the new regenerating tissue, and the functional effects of these signals.Altogether, these experiments will allow us to analyse planarian regeneration in an unprecedented quantitative way and at the single-cell level and with functional information. This will be a significant step towards understanding animal regeneration. Our data will spur novel studies on planarians to study the regulators and processes that we uncover and lead the way in the study of animal regeneration in other animals. The tools and methods that we will use for the first time will become standard in the study of regeneration and developmental processes, and key for single-cell biology. Understanding how animals regenerate is key to advance the agenda of human regenerative medicine.