Mechanisms of specification, quiescence, and regeneration of primordial germ cells

原始生殖细胞的规范、静止和再生机制

• 批准号: 10414946
• 负责人: GARY M WESSEL
• 金额: $ 57.25万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10414946
• 关键词: Adult; Animal Model; Animals; Biochemical; Biological; Biology; Biomedical Research; CRISPR/Cas technology; Cell Lineage; Cell Transplantation; Cells; Cellular biology; Characteristics; Chordata; Complex; Coupled; Development; Drops; Echinodermata; Embryo; Embryonic Development; Evaluation; Event; Female; Gene Expression; Genes; Genetic Transcription; Genomics; Germ Cells; Germ Lines; Heart; Inherited; Knowledge; Mammals; Manuals; Metabolic; Modeling; Molecular; Morphology; Natural regeneration; Oligonucleotides; Organism; Plant Roots; Process; Reagent; Resolution; Sea Urchins; Sexual Reproduction; Signal Pathway; Signal Transduction; Sister; Starfish; Sterility; Structure of primordial sex cell; System; Technology; Tissue Transplantation; Transplantation; Ursidae Family; Vertebrates; Visualization; Work; animal resource; base; chromatin modification; egg; expiration; genetic manipulation; in vivo; innovation; longitudinal analysis; mRNA sequencing; male; optical imaging; optogenetics; regenerative cell; serial imaging; small molecule; sperm cell; stem cells; trait

Project Summary Sexual reproduction requires a germline, a lineage of cells formed in early embryos that ultimately develops into eggs or sperm in the adult. Lore has it that when you lose your germline in development, you become a sterile adult. In many animals, especially in the current favorites of lab animals, that is largely true. We use “rule breakers” though and find such lore unfounded. Our work focuses on the biology of primordial germ cells, how they form during early development, and how they regenerate when the originals are removed. Our work leverages embryos from a sister group to chordates – the sea star and sea urchin. While not common organisms for biomedical research, these echinoderms have many strategic benefits for revealing unique perspectives in the biology of germline formation and regeneration. Millions of synchronous embryos from a single male/female cross allow biochemical and metabolic analysis of the germline, the resultant embryos have ideal transparency for in vivo longitudinal imaging, they develop rapidly, are easy to manipulate (single cell drop-mRNA-seq, optogenetics, cell and tissue transplantations) and they are well suited to complementary gene perturbation approaches (CRISPR/Cas9, morpholinoantisense oligonucleotides, MASO), and small molecule perturbations. The existing deep genomic and reagent resources for these animals, coupled with their tractable experimental characteristics, yields a unique system for understanding primordial germ cell biology with defined molecular and morphological endpoints, in live embryos with longitudinal analysis, distinct metrics of quantitation, and transgenerational evaluations. We interrogate all levels of gene expression for this work, from chromatin modification to post- transcriptional processing and post-translational networks, because that is what the embryos are “telling” us is needed to understand these complex, and deeply rooted events in sexual reproduction. Our work emphasizes longitudinal, in vivo analysis using high resolution optical imaging coupled with genomic perturbations, signal pathway manipulations and manual transplantations and expirations to leverage contrasting mechanisms in germ cell formation between closely related organisms. Sea urchins and sea stars have historically not been genetically manipulated, and this reason is precisely how germ line regeneration has been discovered in this and other animals seen to bear this trait. Relying on manual manipulations meant the genes needed for regeneration were not disturbed, revealing their germ cell regenerative abilities. With new state-of-the-art technologies, these animals can now be exploited with transgenerational analysis. Overall, our work interrogates important biological questions from unique experimental perspectives using rule-breaking models for innovation in the pursuit of new knowledge.

项目摘要 有性繁殖需要生殖系，即在早期胚胎中形成的细胞谱系，最终 在成年后发育成卵子或精子。传说，当你在发育过程中失去生殖系时，你 成为一个不能生育的成年人。在许多动物中，特别是在目前最受欢迎的实验室动物中，这在很大程度上是正确的。 然而，我们使用了“规则破坏者”，并发现这种传说是没有根据的。我们的工作重点是原始生物的生物学 生殖细胞，它们在早期发育过程中是如何形成的，以及当原始细胞被移除时它们是如何再生的。 我们的工作利用了姐妹群的胚胎来脊索动物--海星和海胆。虽然不是 这些棘皮动物是生物医学研究的常见生物，具有许多揭示 在生殖系形成和再生生物学方面的独特视角。数百万个同步胚胎 来自单个雄性/雌性杂交允许对生殖系进行生化和代谢分析，结果 胚胎对体内纵向成像具有理想的透明性，发育迅速，易于操作 (单细胞滴-信使核糖核酸序列，光遗传学，细胞和组织移植)，它们非常适合 互补基因干扰方法(CRISPR/CAS9，吗啉反义寡核苷酸，Maso)， 和小分子扰动。这些动物现有的深层基因组和试剂资源， 再加上它们易于处理的实验特性，产生了一个理解原始生物的独特系统 活体胚胎中具有明确分子和形态终点的生殖细胞生物学 分析、不同的量化指标和跨代评估。 为了这项工作，我们询问了所有水平的基因表达，从染色质修饰到后... 转录处理和翻译后网络，因为这就是胚胎告诉我们的 需要理解有性繁殖中这些复杂而根深蒂固的事件。我们的工作强调 使用结合基因组扰动、信号的高分辨率光学成像进行纵向活体分析 利用对比机制的途径操纵和手动移植和期满 亲缘关系密切的生物体之间的生殖细胞形成。从历史上看，海胆和海星 基因操纵，这就是为什么生殖系再生在这个 以及其他被认为具有这种特征的动物。依靠人工操作意味着需要 再生没有受到干扰，显示了它们的生殖细胞再生能力。拥有最先进的新技术 随着技术的发展，这些动物现在可以通过跨代分析来利用。总体而言，我们的工作 使用打破规则的模型从独特的实验角度询问重要的生物学问题 在追求新知识的过程中进行创新。