Sequential restriction of germ line progenitors by induction

通过诱导连续限制种系祖细胞

• 批准号: 9980947
• 负责人: GARY M WESSEL
• 金额: $ 30.52万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-19 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9980947
• 关键词: Ambystoma; Animal Model; Animals; BMP4; Biochemical; Biological Models; Biomedical Research; Caenorhabditis elegans; Cell Communication; Cells; Characteristics; Chordata; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Coupled; Development; Drops; Drosophila genus; Embryo; Equilibrium; Event; Family member; Female; Fibroblasts; Future; Genes; Genetic Transcription; Genomics; Germ; Germ Cells; Germ Lines; Goals; Gryllidae; Heart; Human; Infertility; Inherited; Mammals; Maps; Messenger RNA; Metabolic; Modeling; Molecular; Morphology; Mus; Nodal; Oocytes; Optics; Organism; PRDM1 gene; Process; RNA; Reagent; Reproduction; Resolution; Resources; Signal Pathway; Signal Transduction; Signaling Molecule; Sister; Somatic Cell; Specific qualifier value; Starfish; Structure of primordial sex cell; System; Technology; Teratoma; Testing; Transforming Growth Factor beta; Translations; Visualization; Work; Xenopus; Zebrafish; cell type; egg; experimental study; gastrulation; implantation; in utero; in vivo; induced pluripotent stem cell; intercellular communication; longitudinal analysis; mRNA sequencing; male; nano; optogenetics; progenitor; response; segregation; serial imaging; single cell mRNA sequencing; small molecule; sperm cell; stem cells; transcription factor; transcriptome; tumor

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.

项目摘要 有性生殖需要一个生殖细胞系，哺乳动物在早期就开始了生殖细胞系的形成。 通过分泌的信号分子来发育。这种体内机制仍然不清楚，主要来自于 胚胎在子宫内发育实验的技术限制。人胚胎中的生殖系诱导 甚至更遥远，过程中的错误与缺乏生殖系（不育）和错误有关。 生殖系细胞（畸胎瘤）的放置规范。原始生殖细胞如何通过细胞间相互作用形成 在早期的发展是本申请的重点，并利用一个姐妹集团脊索动物- 海星星胚胎。 虽然不是生物医学研究的常见有机体，但海星星模型系统具有许多实验性 和揭示这一过程的战略吸引力。来自一个雄性/雌性的数百万个同步胚胎 杂交允许对种系进行生化和代谢分析，得到的胚胎具有理想的 对于体内纵向成像的透明性，它们发展迅速，易于操作（单细胞滴， mRNA-seq，光遗传学对照），并且它们对互补基因扰动方法反应良好 （CRISPR，吗啉代（MASO）和小分子扰动。现有的基因组和试剂资源 对于海星星，再加上海星星胚胎易于处理的实验特性，产生了一个独特的 以及理解哺乳动物生殖系诱导的替代系统。 本应用程序中记录的方法将实现三个主要目标，以推动该领域的发展， 变革的方式。1)三种信号通路优先用于整合刺激和抑制 在早期发展的一个短暂的窗口期间的活动。2)生殖细胞命运限制的机制是 通过限制性内切酶图谱的询问-生殖细胞因子保留在未来的生殖系中，而体细胞因子保留在生殖系中。 来自同一领域的因素迅速受到限制。3)对生殖细胞系的承诺假设包括 与局部胚胎信号迅速隔离，而是转变为具有独特功能的定型细胞类型。 转录组和对信号活性的应答。单细胞mRNA滴测序和光遗传学控制 在单细胞分辨率下的信号传导是通过充分利用光学清晰度和可及性来使用的。 胚胎 总之，这些目标的实现将为理解诱导生殖细胞系提供直接的翻译 形成哺乳动物，并将揭示机制解释生殖细胞少的条件下，导致人类 不育和导致生殖系肿瘤的生殖细胞的错误表达。