Genetic analysis of adult germline stem cell maintenance and homing

描述（由申请人提供）：生殖系干细胞（GSC）对生殖至关重要，保留分化为配子并将遗传信息传递给下一代的能力。GSC依赖于内在和外在信号来归巢到它们的小生境，维持效力，并开始减数分裂以分化成配子。Schlosseri葡萄球菌是一种海洋脊索动物，具有多种特性，使其成为阐明GSC生物学机制的有用和有吸引力的模型。(1)首先，成年葡萄球菌可以在生育和不育阶段循环，同时持续维持GSC的种群。通过比较可育和不育葡萄孢属成虫的全转录组，我们鉴定出了 a TGF？配体，Tgf ²-f，在可育动物中上调，由性腺龛内外的卵泡和卵泡祖细胞表达。在性腺成熟过程中，Tgf？-f在卵母细胞卵泡细胞中同时上调，在睾丸卵泡细胞中下调。我假设TGF-f和TGF？信号传导可能在葡萄球菌GSC的维持、归巢和分化中起作用，我将在本提案的目标1中通过检查TGF β功能丧失动物中的这些过程来验证这一假设。(2)其次，葡萄球菌表现出一种称为生殖细胞寄生（gcp）的现象，其中菌落可以与邻居融合并通过转移GSC取代其种系，这可以通过FACS富集的干细胞群的实验移植来重现。成功替换其融合伴侣的生殖系的集落被称为“赢家”，而生殖系被替换的集落被称为“输家”。“我假设gcp现象是由个体之间GSC增殖和回到生殖细胞龛的能力差异产生的。我进一步假设这些差异存在于GSC内的基因表达或功能水平。我将在本提案的目标2中验证这一假设，方法是从gcp成功者和失败者中分离GSC，并比较其转录组，以确定与gcp成功或失败相关的基因和途径。GSC的失调可以通过导致生殖细胞肿瘤或不育来严重影响人类健康。在这项拟议的研究中要解决的广泛问题是，存在什么机制来确保GSC有效地回到它们的目标生态位，以及一旦到达那里，它们如何在成年生物体中保持未分化状态。解决这些问题将为生殖和再生医学的治疗奠定基础。

DESCRIPTION (provided by applicant): Germline stem cells (GSCs) are critical for reproduction, retaining the capacity to differentiate into gametes and pass genetic information on to the next generation. GSCs depend on both intrinsic and extrinsic signals to home to their niche, maintain potency, and begin meiotic division to differentiate into gametes. Botryllus schlosseri is a marine chordate with several characteristics that make it a useful and attractive model for elucidating the mechanisms of GSC biology. (1) First, adult Botryllus can cycle through phases of fertility and infertility, while continuously maintaining a population of GSCs. By comparing the whole transcriptomes of fertile and infertile Botryllus adults, we have identified a Tgf¿ ligand, Tgf¿-f, upregulated in fertile animals that is expressed by follicle and follicle-progenitor cells both inside and outside of the gonadal niche. Tgf¿-f is also simultaneously upregulated in oocyte follicle cells and downregulated in testicular follicle cells during maturatin of the gonads. I hypothesize that Tgf¿-f and Tgf¿ signaling may play a role in the maintenance, homing, and differentiation of Botryllus GSCs, and I will test this hypothesis in Aim 1 of this proposal by examining these processes in Tgf¿ loss of function animals. (2) Second, Botryllus exhibits a phenomenon called germ cell parasitism (gcp) in which a colony may fuse to a neighbor and replace its germline by the transfer of GSCs, and this can be recapitulated by experimental transplantation of a FACS-enriched stem cell population. Colonies that successfully replace the germline of their fusion partner are referred to as "winners" while those whose germlines are replaced are referred to as "losers." I hypothesize that the phenomenon of gcp is produced by differences between individuals in the ability of their GSCs to proliferate and home to the germ cell niche. I further hypothesize that these differences are present at the level of gene expression or function within GSCs. I will test this hypothesis in Aim 2 of this proposal by FACS-isolating GSCs from gcp winners and losers and comparing their transcriptomes to identify genes and pathways associated with gcp success or failure. Dysregulation of GSCs can severly impact human health by leading to germ cell tumors or infertility. The broad questions being addressed in this proposed study are what mechanisms exist to ensure that GSCs home effectively to their target niche and once there, how they are maintained in an undifferentiated state in the adult organism. Answering these questions will lay the foundation for therapies in reproductive and regenerative medicine.