Ontogeny of the germline in non rodent mammals

非啮齿类哺乳动物种系的个体发育

• 批准号: BB/M001466/1
• 负责人: Ramiro Alberio
• 金额: $ 60.61万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM001466%2F1
• 关键词: Ontogeny; germline; non; rodent; mammals

A better knowledge of how the precursors of the gametes (the cells that make egg and sperm) develop in mammals has important implications for our understanding of animal development, with important applications in biotechnology, assisted reproduction and regenerative medicine.This project will investigate the developmental program of the precursors of the gametes, the primordial germ cells (PGCs), in mammals. Knowledge of mammalian PGCs originates primarily from studies in mice, but very little is known about this process in other mammals. In mice PGCs are set aside very early in development in response to inducing signals from the extraembryonic ectoderm (ExE), a derivative of the trophectoderm. However, the mouse embryo is anatomically different to most other mammals at this stage of development. The mouse embryo (epiblast) undergoes cavitation forming an egg cylinder containing the ExE. In other mammals the ExE does not exist, and the epiblast forms a flat disc of cells. This raises the question of how and when the PGCs originate in humans and other non-rodents mammals lacking the structures (the ExE) known to play a critical role in PGC induction in rodents. We hypothesize that the differences in embryo development underlie differences in the mechanisms of PGC specification in mammals. We will test this hypothesis by investigating PGC development in pig embryos, as they share similar anatomical and developmental features to early human embryos, which are not available for research. Discoveries made in this species can therefore be translated to humans.The aim of this project is to establish the genetic and epigenetic mechanisms of pig PGC development in vivo, followed by functional experiments with cultured isolated epiblasts to identify key master regulators of germ cell development. This knowledge will then be exploited to study human PGC development from human embryonic stem cells (hESC). These investigations will also allow us to determine the relationship between pluripotency and PGC development, which will underpin the development of new culture conditions for the establishment of pluripotent embryonic germ cells. The objectives of the project are: 1- To establish the gene expression profile and epigenetic reprogramming of early pig PGCs. These experiments will define the genetic and epigenetic signature of these cells and will inform on strategies for functional evaluation of PGC determinants. 2- To identify key master regulators that promote PGC differentiation from pig epiblasts and from hESC and pEpiSC/piPSC. These studies will establish the functional role of PGC-master genes during pig (in vivo and in vitro) and human (in vitro) embryonic development, and inform on new strategies for the differentiation of PGC precursors from pluripotent cells. 3- To develop culture conditions for the establishment of pig embryonic germ (EG) cells, the in vitro pluripotent derivatives of PGCs. These experiments will use the knowledge gained from previous objectives to establish pluripotent and germline competent pig EG cells. Insights into primordial germ cell formation will contribute to the development of methods for deriving new sources of pluripotent cells and for improving protocols of differentiation of gamete precursors and somatic cells in the laboratory. An important application of these technologies is in regenerative medicine and assisted reproduction. In addition, new sources of pluripotent cells in domestic animals are of great importance for increasing the efficiency of transgenesis. This project addresses questions of strategic relevance to the BBSRC, such as lifelong health and well-being. We anticipate that the outcomes of the current project will contribute to the academic and clinical advance in the areas of regenerative medicine and global food security.

更好地了解配子前体（产生卵子和精子的细胞）在哺乳动物中如何发育对于我们理解动物发育具有重要意义，并在生物技术、辅助生殖和再生医学中具有重要应用。该项目将研究配子前体，即原始生殖细胞（PGC）在哺乳动物中的发育程序。对哺乳动物 PGC 的了解主要源自对小鼠的研究，但对其他哺乳动物的这一过程知之甚少。在小鼠中，PGC 在发育的早期就被保留下来，以响应来自胚胎外外胚层 (ExE)（滋养外胚层的衍生物）的诱导信号。然而，在这个发育阶段，小鼠胚胎在解剖学上与大多数其他哺乳动物不同。小鼠胚胎（外胚层）经历空化，形成含有 ExE 的卵圆柱体。在其他哺乳动物中，ExE 不存在，并且外胚层形成扁平的细胞盘。这就提出了一个问题：PGCs如何以及何时起源于人类和其他非啮齿类哺乳动物，这些哺乳动物缺乏已知在啮齿类动物PGC诱导中发挥关键作用的结构（ExE）。我们假设胚胎发育的差异是哺乳动物 PGC 规范机制差异的基础。我们将通过研究猪胚胎中 PGC 的发育来检验这一假设，因为它们与早期人类胚胎具有相似的解剖和发育特征，而这些特征无法用于研究。因此，在该物种中取得的发现可以转化为人类。该项目的目的是建立猪 PGC 体内发育的遗传和表观遗传机制，然后用培养的分离外胚层进行功能实验，以确定生殖细胞发育的关键主调节因子。然后，这些知识将被用于研究人类胚胎干细胞 (hESC) 的人类 PGC 发育。这些研究还将使我们能够确定多能性和 PGC 发育之间的关系，这将为建立多能胚胎生殖细胞的新培养条件的开发奠定基础。该项目的目标是： 1- 建立早期猪 PGC 的基因表达谱和表观遗传重编程。这些实验将定义这些细胞的遗传和表观遗传特征，并为 PGC 决定因素的功能评估策略提供信息。 2- 确定促进猪外胚层、hESC 和 pEpiSC/piPSC 中 PGC 分化的关键主调节因子。这些研究将确定 PGC 主基因在猪（体内和体外）和人类（体外）胚胎发育过程中的功能作用，并为从多能细胞中分化出 PGC 前体的新策略提供信息。 3- 开发建立猪胚胎生殖（EG）细胞（PGC 的体外多能衍生物）的培养条件。这些实验将利用从先前目标中获得的知识来建立多能和种系感受态猪 EG 细胞。对原始生殖细胞形成的深入了解将有助于开发多能细胞新来源的方法，并改进实验室中配子前体和体细胞的分化方案。这些技术的一个重要应用是再生医学和辅助生殖。此外，家畜多能细胞的新来源对于提高转基因效率具有重要意义。该项目解决与 BBSRC 具有战略相关性的问题，例如终身健康和福祉。我们预计当前项目的成果将有助于再生医学和全球粮食安全领域的学术和临床进步。

项目成果

Simulating gastrulation development and germ cell fate in vitro using human and monkey pluripotent stem cells

使用人和猴多能干细胞在体外模拟原肠胚发育和生殖细胞命运

• DOI: 10.1038/protex.2017.050
• 发表时间: 2017
• 期刊: Protocol Exchange
• 作者: Kobayashi T

Lineage segregation, pluripotency and X-chromosome inactivation in the pig pre-gastrulation embryo

• DOI: 10.1101/347823
• 发表时间: 2018-06
• 期刊: bioRxiv
• 作者: P. Ramos-Ibeas;Fei Sang;Qifan Zhu;Walfred W. C. Tang;Sarah Withey;Doris Klisch;M. Loose;M. Surani;R. Alberio

Pluripotency and X chromosome dynamics revealed in pig pre-gastrulating embryos by single cell analysis.

通过单细胞分析揭示猪原肠胚形成前胚胎的多能性和 X 染色体动力学。

• DOI: 10.17863/cam.38551
• 发表时间: 2019
• 作者: Ramos-Ibeas P

Principles of early human development and germ cell program from observed model systems

来自观察模型系统的早期人类发育和生殖细胞程序的原理

• DOI: 10.17863/cam.10360
• 发表时间: 2017
• 作者: Kobayashi T

Primordial germ cells: the first cell lineage or the last cells standing?

• DOI: 10.1242/dev.113993
• 发表时间: 2015-08-15
• 期刊: Development (Cambridge, England)
• 作者: Johnson AD;Alberio R
• 通讯作者: Alberio R