Germ cell specification and differentiation in planarians

项目总结/摘要 生殖细胞产生配子-卵母细胞和精子-融合产生全能合子，合子分裂并 生成一个有机体的所有细胞。在发育过程中，必须建立新的生殖细胞谱系， 正确区分，以确保物种的繁殖。生殖细胞特化的大部分知识， 发育来自模式生物，在模式生物中，母体决定因素在发育早期指定生殖细胞。 胚胎发生相比之下，许多动物（包括哺乳动物）对生殖细胞的反应是： 在发育后期来自周围细胞的诱导信号。尽管这些差异在生殖系 建立时，所有生殖细胞都具有几个重要的特征，如内源性转录抑制， 体细胞程序和对来自体细胞支持细胞的外在因素的依赖。自由生活 扁形虫，Schmidtea meditangleea，以其非凡的再生能力而闻名， 依赖于一群多能性体干细胞。真涡虫在胚胎期后就确定了它们的生殖细胞 从这些体细胞干细胞中分离出来。引人注目的是，真涡虫甚至可以再生新的 生殖细胞新生过去十年的工作已经确立了涡虫作为一个易于处理的模型， 研究生殖细胞生物学，以及调节男性不同方面的几个内在和外在因素。 已经确定了生殖系发育-规格、维持和分化。但几乎 对这些动物的雌性生殖细胞发育一无所知，这是一个关键的空白，因为重要的是 寄生性扁形虫病的病理学中卵产生的作用。在这里，建立在广泛的初步 根据来自申请人实验室的数据，提出了以下两个目标：（i）在功能上表征 卵巢转录组;（ii）检查单胺神经递质在生殖细胞中的作用 发展在目标1中，激光捕获显微切割和下一代RNA测序将用于 创造出富含卵巢和睾丸的转录组通过原位杂交验证卵巢富集表达 通过RNA干扰来表征基因功能，将能够鉴定生殖细胞内在的， 女性生殖细胞发育的外在调节因子。在Aim 2中，基于尖端质谱的 代谢组学研究将与基因表达图谱和功能方法相结合， 生物源性单胺，其局部和/或系统地发出信号以调节生殖细胞发育。的 拟议中的实验利用了涡虫的再生能力和功能基因组工具， 可用于研究这些动物，以全面了解调节雌性生殖细胞的基因 发展这些研究是有意义的，因为它们有望提高对生殖细胞的理解。 研究后生动物的生物学，并帮助指导未来对哺乳动物系统的实验。另外这款 这项工作将为破坏寄生扁虫的繁殖提供新的靶点，寄生扁虫利用其 大量的生殖产出将传播并影响全世界数亿人。

Project Summary/Abstract Germ cells give rise to gametes–oocytes and sperm–that fuse to create a totipotent zygote, which divides and generates all the cells of an organism. During development, a new germ cell lineage must be established and differentiate properly to ensure propagation of the species. Most knowledge of germ cell specification and development comes from model organisms in which maternal determinants specify germ cells early in embryogenesis. By contrast, many animals (including mammals) specify their germ cells in response to inductive cues from surrounding cells later in development. Despite these differences in germline establishment, all germ cells share several important features, such as intrinsic transcriptional repression of somatic cell programs and dependence on extrinsic factors from somatic support cells. The free-living planarian flatworm, Schmidtea mediterranea, is well known for its extraordinary regenerative prowess, which relies on a population of pluripotent somatic stem cells. Planarians specify their germ cells post-embryonically from these somatic stem cells in response to inductive cues. Strikingly, planarians can even regenerate new germ cells de novo. Work over the past decade has established the planarian as a tractable model in which to study germ cell biology, and several intrinsic and extrinsic factors that regulate distinct aspects of male germline development–specification, maintenance, and differentiation–have been identified. However, almost nothing is known about female germ cell development in these animals, a critical gap because of the important role of egg production in the pathology of parasitic flatworm diseases. Here, building on extensive preliminary data from the applicant’s laboratory, the following two aims are proposed: (i) to functionally characterize the ovarian transcriptome; and (ii) to examine the role(s) of monoamine neurotransmitters in germ cell development. In Aim 1, laser capture microdissection and next-generation RNA sequencing will be used to create ovary- and testis-enriched transcriptomes. Validating ovary-enriched expression by in situ hybridization and characterizing gene function by RNA interference will enable the identification of germ cell-intrinsic and extrinsic regulators of female germ cell development. In Aim 2, cutting-edge mass spectrometry-based metabolomic studies will be combined with gene expression mapping and functional approaches to investigate the biogenic monoamines that signal locally and/or systemically to regulate germ cell development. The proposed experiments capitalize upon the planarian’s regenerative abilities and the functional genomic tools available for studying these animals to acquire a comprehensive view of the genes regulating female germ cell development. These studies are significant as they are expected to enhance understanding of germ cell biology across the metazoans and help guide future experiments on mammalian systems. In addition, this work will provide novel targets for disrupting reproduction in parasitic flatworms, which capitalize on their prolific reproductive output to propagate and afflict hundreds of millions of people worldwide.