Ontogeny of the germline in non rodent mammals

更好地了解哺乳动物配子的前体（制造卵子和精子的细胞）是如何发育的，对我们理解动物发育有着重要的意义，在生物技术、辅助生殖和再生医学方面有着重要的应用。本项目将研究哺乳动物配子前体——原始生殖细胞（PGCs）的发育过程。对哺乳动物PGCs的了解主要来自对小鼠的研究，但对其他哺乳动物的这一过程知之甚少。在小鼠中，PGCs在发育的早期就被放置在一边，以响应来自胚外外胚层（一种滋养外胚层的衍生物）的诱导信号。然而，在这个发育阶段，老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的卵筒。在其他哺乳动物中，ExE不存在，外胚层形成扁平的圆盘状细胞。这就提出了PGCs如何以及何时在人类和其他非啮齿动物哺乳动物中产生的问题，这些哺乳动物缺乏已知在啮齿动物的PGC诱导中起关键作用的结构（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和发育特征，而这些特征尚无法用于研究。因此，在这个物种中取得的发现可以转化为人类。本项目旨在建立猪PGC在体内发育的遗传和表观遗传机制，并在体外培养的离体外胚层进行功能实验，以确定生殖细胞发育的关键主控调控因子。这些知识将被用于研究人类胚胎干细胞（hESC）的PGC发育。这些研究也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发展奠定基础。本项目的目标是：1-建立早期猪PGCs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决定因素的功能评估策略提供信息。2-确定促进PGC从猪外表皮细胞、hESC和pEpiSC/piPSC分化的关键主调控因子。这些研究将确定PGC主基因在猪（体内和体外）和人（体外）胚胎发育中的功能作用，并为PGC前体从多能细胞分化提供新的策略。3-为猪胚胚细胞（PGCs）体外多能衍生物)的培养条件研究。这些实验将利用从以前的目标中获得的知识来建立多能性和种系能力的猪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，并有助于改进配子前体和体细胞在实验室中的分化方案。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提高转基因效率具有重要意义。该项目解决了与BBSRC战略相关的问题，例如终身健康和福祉。我们期待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.