Cellular and Molecular Mechanisms of Germline Regeneration

项目总结 人类和成熟的研究机构缺乏再生生殖细胞(生殖细胞)的能力 细胞)和生殖器官。来自这些生物体的研究结果确立了目前的观点，即细菌 细胞是与胞体分离的不同谱系；因此，生殖细胞的丧失使生物体不育。 因为新的生殖细胞不能从胞体中衍生出来。与这一被广泛接受的观点相矛盾的是， 许多生物(如九头蛇、扁虫、节虫和海星)都能很容易地再生细菌 细胞。然而，人们对这些生物中再生生殖细胞的细胞来源知之甚少。这个 我的实验室的目标是弥合这一知识鸿沟，并确定细胞起源和分子 生殖细胞再生的机制。使用现有的研究机构来解决这个问题是不可行的 像老鼠、果蝇和线虫一样，因为它们不能再生生殖细胞。此外，许多 能够再生生殖细胞的生物不利于研究这一过程的机制，因为 他们缺乏转基因工具，或者他们的解剖结构带来了技术挑战，比如大而不透明的身体，或者 无法接触到生殖器官。这些限制阻碍了实时成像的使用--实时成像是跟踪 生殖细胞及其来源细胞的谱系。我们使用一种节段性的蠕虫--扁平线虫--来研究细菌 细胞再生。扁豆非常适合于这项研究，因为生殖细胞再生可以被诱导并被 快速实现；转录组数据库、基因组草图和转基因工具(对遗传谱系至关重要 可供选择)；小巧而透明的机身使其非常适合实时成像。因此， Platynereis是一种研究有机体，它为结合现代技术提供了难得的机会 需要研究生殖细胞再生(活体成像、遗传谱系追踪、转录学) 相关的胚胎后生命阶段(即青少年、成人)，这些阶段通常对生活的想象具有挑战性。 我们假设再生生殖细胞的细胞来源有三种可能的模式：1)多能/多能 干细胞可以再生体细胞和生殖细胞；2)血统受限的细胞群体致力于 只再生生殖细胞；或3)体细胞通过重新编程转分化为生殖细胞。我们的 实验方法将是：a)测试三种生殖细胞模型之间的生殖细胞再生通过基因 细胞谱系追踪和实时成像。这将使我们能够确定产生细菌的确切细胞谱系。 细胞在发育和再生过程中。B)确定在重新编程期间发生的分子变化 通过单细胞RNA测序将来源细胞转化为生殖细胞。这将使我们能够获得转录组的轨迹 在再生过程中并识别源细胞中的细胞类型特定标记，中间状态， 和新的生殖细胞。这些标记将在未来的研究中进行功能测试。该项目将显著地 有助于我们对生殖细胞生物学和体细胞-生殖细胞区别的基本理解。

PROJECT SUMMARY Humans and well-established research organisms lack the ability to regenerate their reproductive cells (germ cells) and reproductive organs. Research findings from these organisms established the current view that germ cells are a distinct lineage separated from the soma; therefore, the loss of germ cells renders an organism infertile because new germ cells cannot be derived from the soma. Contradicting this widely accepted view is the fact that many organisms (e.g. hydra, flatworms, segmented worms, and sea stars) can readily regenerate germ cells. However, the cellular source of regenerated germ cells in these organisms is very poorly understood. The goal of my laboratory is to close this knowledge gap and define the cellular origins and molecular mechanisms of germ cell regeneration. Addressing this is not feasible using established research organisms like mice, fruit flies, and nematodes, because they do not regenerate germ cells. Furthermore, many of the organisms that can regenerate germ cells are not conducive to studying the mechanisms of this process because they lack transgenic tools, or their anatomies present technical challenges such as large and opaque bodies, or inaccessibility of reproductive organs. These limitations prevent the use of live imaging – a key tool to trace the lineages of germ cells and their source cells. We use a segmented worm, Platynereis dumerilii, for studying germ cell regeneration. Platynereis is well-suited for this study because germ cell regeneration can be induced and is achieved quickly; transcriptome databases, a draft genome, and transgenic tools (critical for genetic lineage tracing) are available; and a small and transparent body makes it excellent for live imaging. Therefore, Platynereis is a research organism that presents a rare opportunity to combine the modern techniques required to study germ cell regeneration (live-imaging, genetic lineage tracing, transcriptomics) in the relevant post-embryonic life stages (i.e. juveniles, adults) which are typically challenging to image live. We postulate three possible models for the cellular sources for the regenerated germ cells: 1) pluri/multipotent stem cells regenerate both somatic and germ cells; 2) a lineage-restricted cell population is dedicated to regenerating only the germ cells; or 3) somatic cells transdifferentiate into germ cells by reprogramming. Our experimental approach will be to: a) Test between the three cellular models of germ cell regeneration via genetic cell lineage tracing and live imaging. This will allow us to identify the exact cell lineages that give rise to germ cells during development and regeneration. b) Identify the molecular changes taking place during reprogramming source cells into germ cells by single cell RNA sequencing. This will allow us to obtain transcriptome trajectories over the course of regeneration and identify cell type-specific markers in the source cells, the intermediate states, and the new germ cells. These markers will be tested for function in future studies. The project will significantly contribute to our fundamental understanding of germ cell biology and the soma-germ cell distinction.