Cellular and Molecular Mechanisms of Germline Regeneration

种系再生的细胞和分子机制

• 批准号: 10027827
• 负责人: Busra Duygu Ozpolat
• 金额: $ 40.18万
• 依托单位: MARINE BIOLOGICAL LABORATORY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10027827
• 关键词: Address; Adolescent; Adult; Anatomy; Animals; Annelida; Cell Lineage; Cell model; Cells; Cellular biology; Databases; Development; Drosophila genus; Embryo; Future; Genes; Genetic; Genome; Germ Cells; Goals; Human; Image; Infertility; Knowledge; Laboratories; Life; Modernization; Molecular; Multipotent Stem Cells; Mus; Natural regeneration; Nematoda; Organism; Platyhelminths; Population; Process; Regenerative Medicine; Research; Somatic Cell; Source; Starfish; Techniques; Testing; Transgenic Organisms; cell regeneration; cell type; egg; imaging genetics; neuronal cell body; prevent; reproductive organ; single-cell RNA sequencing; sperm cell; stem cell biology; stem cells; tissue regeneration; tool; transcriptome; transcriptomics

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.

项目摘要 人类和建立的研究生物缺乏再生其生殖细胞的能力（胚芽 细胞）和生殖器官。这些生物体的研究结果确定了当前的观点 细胞是与躯体分离的独特谱系。因此，生殖细胞的损失使生物不育 因为新生殖细胞不能源自躯体。与这种广泛接受的观点相矛盾是事实 许多生物（例如九头蛇，扁虫，分段蠕虫和海星）可以容易再生胚芽 细胞。然而，这些生物中再生生殖细胞的细胞来源非常了解。这 我的实验室的目标是缩小此知识差距并定义细胞起源和分子 生殖细胞再生的机制。使用已建立的研究生物来解决这是不可行的 像小鼠，果蝇和线虫一样，因为它们不会再生生殖细胞。此外，许多 可以再生生殖细胞的生物没有引导性研究此过程的机制，因为 他们缺乏转基因工具或其解剖学提出的技术挑战，例如大型和不透明的身体，或者 生殖器官的无法访问。这些限制阻止了实时成像的使用 - 追踪的关键工具 生殖细胞及其源细胞的谱系。我们使用分段的蠕虫dumerilii来研究胚芽 细胞再生。铂藻非常适合这项研究，因为可以诱导生殖细胞再生，并且是 迅速实现；转录组数据库，基因组草案和转基因工具（遗传谱系至关重要） 跟踪）可用；一个小而透明的身体使其非常适合现场成像。所以， Platyneris是一种研究生物，它提供了一个难得的结合现代技术的机会 需要研究生殖细胞再生（现场模仿，遗传谱系追踪，转录组学） 相关的胚胎后生活阶段（即少年，成年人）通常会挑战现场图像。 我们假设了再生生殖细胞的细胞源的三个可能的模型：1）pluri/多能 干细胞再生体细胞和生殖细胞。 2）谱系限制的细胞种群专用于 仅再生生殖细胞；或3）通过重编程将体细胞转分化为生殖细胞。我们的 实验方法将是：a）生殖细胞再生的三个细胞模型通过遗传测试 细胞谱系跟踪和现场成像。这将使我们能够确定引起细菌的确切细胞谱系 细胞在发育和再生过程中。 b）确定重新编程过程中发生的分子变化 源细胞通过单细胞RNA测序进入生殖细胞。这将使我们能够获得转录组轨迹 在再生过程中，并确定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。这些标记将在以后的研究中测试功能。该项目将大大 有助于我们对生殖细胞生物学和Soma-Germ细胞区别的基本理解。