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: 10631065
• 负责人: GARY M WESSEL
• 金额: $ 57.27万
• 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/10631065
• 关键词: Adult; 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; Genomics; Germ Cells; Germ Lines; Heart; Inherited; Knowledge; Mammals; Manuals; Metabolic; Modeling; Molecular; Morphology; Natural regeneration; Oligonucleotides; Organism; Process; Reagent; Resolution; Sea Urchins; Sexual Reproduction; Signal Pathway; Signal Transduction; Sister; Specific qualifier value; Starfish; Sterility; Structure of primordial sex cell; System; Technology; Tissue Transplantation; Transplantation; Vertebrates; Visualization; Work; animal resource; chromatin modification; egg; expiration; genetic manipulation; in vivo; innovation; longitudinal analysis; mRNA sequencing; male; model organism; optical imaging; optogenetics; posttranscriptional; 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 有性生殖需要一个生殖系，一个在早期胚胎中形成的细胞谱系， 在成虫中发育成卵子或精子。传说中，当你在发育过程中失去生殖细胞时， 成为不育的成年人。在许多动物中，尤其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。 我们使用“规则破坏者”，但发现这样的传说毫无根据。我们的工作集中在原始的生物学 生殖细胞，它们在早期发育过程中如何形成，以及当原始细胞被移除时它们如何再生。 我们的工作利用了脊索动物姐妹群的胚胎--海星星星和海胆。虽然不 生物医学研究的常见生物，这些棘皮动物有许多战略利益，揭示 在生殖系形成和再生生物学的独特观点。数以百万计的同步胚胎 允许对种系进行生化和代谢分析， 胚胎具有用于体内纵向成像的理想透明度，它们发育迅速，易于操作， （单细胞滴-mRNA-seq，光遗传学，细胞和组织移植），它们非常适合 互补基因扰动方法（CRISPR/Cas9，吗啉代反义寡核苷酸，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，产生了一个独特的系统，用于理解原始的 生殖细胞生物学，具有明确的分子和形态学终点，在活胚胎中， 分析，不同的量化指标，以及跨代评估。 我们询问所有水平的基因表达这项工作，从染色质修饰后， 转录处理和翻译后网络，因为这就是胚胎“告诉”我们的。 需要了解这些复杂的，根深蒂固的事件在有性生殖。我们的工作强调 使用高分辨率光学成像结合基因组扰动的纵向体内分析， 途径操作和手工移植和移植，以利用 生殖细胞的形成在密切相关的生物体之间。海胆和海星在历史上 基因操纵，而这个原因正是如何在这个过程中发现了生殖系再生。 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基因需要 再生没有受到干扰，揭示了它们的生殖细胞再生能力。拥有最先进的 通过技术，这些动物现在可以通过跨代分析来利用。总的来说，我们的工作 从独特的实验角度使用打破规则的模型来询问重要的生物学问题 在追求新知识的过程中进行创新。