Ontogeny of the germline in non rodent mammals

本项目将研究哺乳动物配子前体细胞（原始生殖细胞）的发育过程，为进一步了解哺乳动物配子前体细胞的发育过程提供科学依据。本项目将研究哺乳动物配子前体细胞（原始生殖细胞）的发育过程。哺乳动物PGCs的知识主要来源于对小鼠的研究，但对其他哺乳动物的这一过程知之甚少。在小鼠中，PGCs在发育的非常早期被搁置，以响应来自胚外外胚层（ExE）（滋养外胚层的衍生物）的诱导信号。然而，小鼠胚胎在发育的这个阶段与大多数其他哺乳动物在解剖学上不同。小鼠胚胎（外胚层）经历空化，形成含有ExE的卵柱。在其他哺乳动物中，ExE不存在，上胚层形成一个扁平的细胞盘。这就提出了一个问题，即PGC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中PGC诱导中起关键作用的结构（ExE）。我们推测，胚胎发育的差异是哺乳动物PGC特化机制差异的基础。我们将通过研究猪胚胎中的PGC发育来验证这一假设，因为它们与早期人类胚胎具有相似的解剖学和发育特征，而这些特征无法用于研究。该项目的目的是建立猪PGC在体内发育的遗传和表观遗传机制，然后通过培养分离的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。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，这将为建立多能性胚胎生殖细胞的新培养条件的开发奠定基础。本研究的主要目的是：1-建立猪早期原始生殖细胞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，并将为PGC决定簇的功能评估策略提供信息。2-鉴定促进PGC从猪外胚层和从hESC和pEpiSC/piPSC分化的关键主调节因子。这些研究将建立PGC-主基因在猪（体内和体外）和人（体外）胚胎发育过程中的功能作用，并为从多能细胞分化PGC前体的新策略提供信息。3-建立猪胚胎生殖细胞（EG细胞）的体外培养条件。这些实验将使用从先前目的获得的知识来建立多能和生殖系感受态猪EG细胞。对原始生殖细胞形成的深入了解将有助于开发用于获得多能细胞的新来源的方法，以及改进实验室中配子前体细胞和体细胞分化的方案。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。该项目涉及与BBSRC具有战略意义的问题，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。

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.