Cellular and Molecular Mechanisms of Germline Regeneration

项目摘要 人类和成熟的研究生物缺乏再生生殖细胞（生殖细胞）的能力 细胞和生殖器官。对这些生物体的研究结果确立了目前的观点， 生殖细胞是从索马中分离出来的一个独特的谱系;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索马中产生。与这一被广泛接受的观点相矛盾的是， 许多生物体（如水螅、扁形虫、分节蠕虫和海星）可以很容易地再生细菌， 细胞然而，这些生物体中再生生殖细胞的细胞来源知之甚少。的 我的实验室的目标是缩小这一知识差距，并确定细胞起源和分子 生殖细胞再生的机制。使用已建立的研究生物体解决这一问题是不可行的 比如老鼠、果蝇和线虫，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体不利于研究这一过程的机制， 它们缺乏转基因工具，或者它们的解剖结构带来了技术挑战，例如巨大而不透明的身体，或者 生殖器官无法进入。这些限制阻止了实时成像的使用，实时成像是追踪 生殖细胞及其来源细胞的谱系。我们用一种分节的蠕虫，Platynereis dumerilii， 细胞再生Platynereis非常适合这项研究，因为生殖细胞再生可以被诱导， 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 跟踪）;和一个小而透明的机构，使其非常适合现场成像。因此，我们认为， Platynereis是一种研究生物，它提供了一个难得的机会，联合收割机的现代技术 需要研究生殖细胞再生（活成像，遗传谱系追踪，转录组学）， 相关的胚胎后生命阶段（即青少年，成年人），这通常是具有挑战性的图像生活。 我们提出了三种可能的再生生殖细胞的细胞来源模型：1）多能/多能 干细胞再生体细胞和生殖细胞; 2）谱系限制的细胞群致力于 仅再生生殖细胞;或3）体细胞通过重编程转分化成生殖细胞。我们 实验方法将是：a）通过遗传学方法在生殖细胞再生的三种细胞模型之间进行测试， 细胞谱系追踪和活体成像。这将使我们能够确定产生生殖细胞的确切细胞谱系 细胞的发育和再生。B）鉴定重编程期间发生的分子变化 通过单细胞RNA测序将细胞源转化为生殖细胞。这将使我们能够获得转录组轨迹 在再生过程中，识别源细胞中的细胞类型特异性标记，中间状态， 和新的生殖细胞这些标记物将在未来的研究中进行功能测试。该项目将大大 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.