Germ cell specifications and differentiation in planarians

描述(申请人提供)：生殖细胞作为世代之间的纽带，产生配子，将遗传物质从父母传给后代。了解生殖细胞发育的机制将有助于阐明全能的本质、不孕不育的原因以及生殖细胞肿瘤的起源。生殖细胞发育的许多知识来自对模型生物的研究，这些模型生物利用细胞质决定因素来确定早期胚胎发育中生殖细胞的命运。相反，许多生物(包括哺乳动物)利用感应信号来确定生殖细胞在发育后期的命运。尽管有这些差异，但由决定因素指定的生殖细胞和那些诱导形成的生殖细胞有几个共同的内在机制，包括转录抑制体细胞命运和转录后控制基因表达。诱导性生殖细胞发育的机制研究几乎完全局限于小鼠，重要的问题仍然没有答案。例如，什么样的转录网络定义和维持生殖细胞的命运？在指定生殖细胞后，哪些系统因素将其进一步分化与动物的生理状态联系起来？淡水浮游生物，米德米特里亚，是研究这些问题的一个强有力的模型。它具有惊人的再生能力，基于成体干细胞群体，即使是微小的身体碎片也能再生出完整的个体。在浮游生物中，生殖细胞谱系从体细胞干细胞在胚胎后发育而来；像体细胞一样，生殖细胞谱系也可以再生。申请人实验室产生的初步数据已经确定了维持生殖细胞和调节其分化所需的内在和外在调节因子。基于这些数据，将追求两个特定的目标：(I)表征早期生殖细胞的转录网络；(Ii)剖析以神经肽为基础的生殖细胞分化的系统调节。 在第一个目标下进行的工作将使用染色质免疫沉淀、下一代测序和计算方法来识别维持早期生殖细胞所需的生殖细胞特异性转录因子的靶标。在目标二下进行的实验将表征介导生殖细胞发育的系统调控的神经肽受体，鉴定表达该受体的细胞类型(S)，并发现响应神经肽信号的基因。为了达到这两个目的，将使用研究地中海链霉菌的功能基因组学工具(高通量原位杂交；通过喂食双链RNA进行RNA干扰)来验证目标基因并确定其功能。这些研究使用具有独特属性的模式生物，并采用无偏见的基因组方法来确定保守因素；因此，它们具有巨大的创新潜力。这项工作意义重大，因为它可能：(I)识别生殖细胞正常发育所需的保守基因；以及(Ii)为理解和治疗感染全球数亿人的寄生虫提供一种有前途的策略。

DESCRIPTION (provided by applicant): Germ cells serve as a link between generations, producing gametes that propagate genetic material from parent to offspring. Understanding the mechanisms by which germ cells develop will help illuminate the nature of totipotency, the causes of infertility, and the origins of germ cell tumors. Much knowledge of germ cell development derives from studying model organisms that utilize cytoplasmic determinants to specify germ cell fate in early embryogenesis. In contrast, many organisms (including mammals) utilize inductive signals to specify germ cell fate later in development. In spite of these differences, germ cells specified by determinants and those formed inductively share several common intrinsic mechanisms, including transcriptional repression of somatic fates and post-transcriptional control of gene expression. Mechanistic studies of inductive germ cell development have been limited almost exclusively to mouse and important questions remain unanswered. For example, what transcriptional networks define and maintain germ cell fate? After a germ cell has been specified, what systemic factors link its further differentiation with te physiological status of the animal? The freshwater planarian, Schmidtea mediterranea, serves as a powerful model for studying these questions. It has prodigious regenerative abilities, based upon a population of adult stem cells that allow even tiny body fragments to regenerate complete individuals. In planarians, the germ cell lineage develops post- embryonically from the somatic stem cells; like the soma, the germ cell lineage can also be regenerated. Preliminary data generated in the applicant's laboratory have identified intrinsic and extrinsic regulators required for maintaining germ cells and for regulating their differentiation. Based on these data, two specific aims will be pursued: (i) to characterize the transcriptional network in early germ cells; and (ii) to dissect the neuropeptide-based systemic regulation of germ cell differentiation. Work performed under the first aim will use chromatin immuno-precipitation, next-generation sequencing, and computational approaches to identify targets of a germ cell-specific transcription factor required for maintaining early germ cells. Experiments conducted under aim two will characterize the neuropeptide receptor mediating the systemic regulation of germ cell development, identify the cell type(s) that express the receptor, and discover genes expressed in response to neuropeptide signaling. For both aims, the functional genomic tools for studying S. mediterranea (high- throughput in situ hybridization; RNA interference by feeding double-stranded RNA) will be used to validate target genes and determine their functions. These studies use a model organism with unique attributes, and take unbiased, genomic approaches to identify conserved factors; thus, they have great potential to innovate. This work is significan because of its potential: (i) to identify conserved genes required for proper germ cell development; and (ii) to provide a promising strategy for understanding and treating parasitic flatworms that infect hundreds of millions of people worldwide.