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

项目摘要 我们请求通过这一行政补充机制为一个冰鲜海洋水族馆单元提供支持，以 我们的几个棘皮动物物种，以及我们从实验中获得的基因操纵的成虫 在父应用程序中建议。我们成立16年的主要海洋水族馆单位已经失败，我们的工作已经 大大超出了它的能力，在这里我们请求支持，用一个能够为我们提供 功能和扩展的容量。这项请求支持的研究重点是了解 胚胎发生过程中原始生殖细胞的形成机制及其早期形成机制 发展，以及当原件被移除时它们如何再生。我们的工作利用了来自 脊索动物的姐妹群--海星和海胆。虽然不是生物医学研究的常见生物， 这些棘皮动物在揭示生殖系生物学的独特视角方面有许多战略上的好处。 形成和再生。来自单个雄性/雌性杂交的数百万个同步胚胎允许 对胚系进行生化和代谢分析，得到的胚胎具有理想的体内透明性 它们发展迅速的纵向成像很容易操作(单细胞下降-mRNA-SEQ，光遗传学， 细胞和组织移植)，它们非常适合于互补基因扰动方法 (CRISPR/CAS9，吗啉反义寡核苷酸，MISO)和小分子扰动。现有的 这些动物的深层基因组和试剂资源，加上它们易于处理的实验 特征，为理解原始生殖细胞生物学提供了一个独特的系统，并定义了分子 和形态终点，在活胚胎中进行纵向分析，不同的量化指标，以及 跨代评估。

Project Summary We request support through this administrative supplement mechanism for a chilled marine aquarium unit to house our several echinoderm species, and our genetically manipulated adults derived from the experiments proposed in the parent application. Our 16-year-old main marine aquarium unit has failed, and our work has significantly expanded beyond its capacity and here we request support to replace it with a unit that will give us functionality and expanded capacity. The research supported by this request is focused on understanding the mechanisms of formation of primordial germ cells during embryogenesis, 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.