Sequential restriction of germ line progenitors by induction

项目摘要 有性繁殖需要一个生殖系，哺乳动物在早期就开始了它们的生殖系 通过分泌的信号分子发育。体内的这种机制仍然不清楚，主要是因为 对子宫内发育的胚胎进行实验的技术限制。人类胚胎生殖系的诱导 甚至更遥远，过程中的错误与缺乏生殖系(不育)和错配有关。 放置生殖系细胞(畸胎瘤)的规格。原始生殖细胞是如何通过细胞间的相互作用形成的 在早期开发中是这个应用程序的重点，并利用姐妹组来协调 海星胚胎。 虽然不是生物医学研究的常见生物，但海星模型系统有许多实验 以及揭示这一过程的战略吸引力。来自单个雄性/雌性的数百万个同步胚胎 杂交允许对胚系进行生化和代谢分析，所得到的胚胎具有理想的 体内纵向成像的透明度，发展迅速，易于操作(单细胞滴- Mrna-seq，光遗传控制)，它们对互补基因扰动方法反应良好。 (CRISPR、吗啉(Maso)和小分子扰动)。现有的基因组和试剂资源 对于海星来说，再加上海星胚胎易驯服的实验特性，产生了独特的 以及用于理解哺乳动物生殖系诱导的代理系统。 本应用程序中记录的方法将实现三个主要目标，以在 变革性的方式。1)三条信号通路优先进行整合刺激和抑制 在早期发展的短暂窗口内的活动。2)生殖细胞命运限制的机制是 被限制图询问-生殖细胞因子被保留在未来的生殖系中，而体细胞 同一领域的因素很快就会受到制约。3)对生殖系的承诺假设包括 快速隔离局部胚胎信号，而不是过渡到具有唯一 转录组，以及对信号活动的反应。单细胞信使核糖核酸滴定与光遗传调控 通过充分利用光学清晰度和可访问性来使用单小区分辨率的信令 胚胎。 总之，这些目标的实现将为理解诱导胚系提供直接的翻译 在哺乳动物中形成，并将揭示导致人类无生殖细胞条件的机制解释 不孕不育和生殖细胞错误表达导致生殖系肿瘤。

Project Summary Sexual reproduction requires a germ line and mammals craft the beginning of their germ line early in development by secreted signaling molecules. This mechanism in vivo remains obscure, largely from the technical limitations of experimenting on embryos developing in utero. Germ line induction in a human embryo is even more remote and errors in the process are associated with a lack of germ line (infertility) and mis- placed specification of germ line cells (teratomas). How the primordial germ cells form by cell-cell interactions during early development is the focus of this application and makes use of a sister group to chordates – the sea star embryo. While not a common organism for biomedical research, the sea star model system has many experimental and strategic attractions for revealing this process. Millions of synchronous embryos from a single male/female cross allow biochemical and metabolic analysis of the germ line, the resultant embryos have ideal transparency for in vivo longitudinal imaging, they develop rapidly, are easy to manipulate (single cell drop- mRNA-seq, optogenetic controls) and they respond well to complementary gene perturbation approaches (CRISPR, morpholino (MASO), and small molecule perturbations. The existing genomic and reagent resources for the sea star, coupled with the tractable experimental characteristics of the sea star embryo, yields a unique and surrogate system for understanding mammalian germ line induction. The approaches documented in this application will accomplish three main goals to advance the field in transformative ways. 1) Three signaling pathways are prioritized for integrative stimulating and repressing activities during a brief window in early development. 2) The mechanism of germ cell fate restriction is interrogated by a restriction map - germ cell factors are retained in the future germ line, whereas somatic factors are rapidly restricted from the same field. 3) Commitment to the germ line is hypothesized to include rapid insulation from local embryonic signaling and instead transitions to a committed cell type with a unique transcriptome, and response to signal activity. Single cell mRNA drop-sequencing and optogenetic control over signaling at single cell resolution are used by taking full advantage of the optical clarity and accessibility of the embryo. Overall, these accomplished goals will provide direct translation for understanding inductive germ line formation in mammals, and will reveal mechanistic explanations for germ cell-less conditions leading to human infertility, and mis-expression of germ cells leading to germ line tumors.