Mechanisms of Sex Determination in Zebrafish

正常的人类性腺发育在子宫内会受到未知的遗传和环境因素的不利影响，导致睾丸发育障碍综合征和多囊卵巢综合征。在哺乳动物中，y染色体基因SRY启动了一个尚不完全了解的遗传网络，该网络指导性腺成为睾丸，而竞争基因指导卵巢发育。非哺乳类脊椎动物也使用类似的下游网络，但主要性别决定（SD）基因不同。此外，在某些物种中，温度或营养等环境因素会影响SD。我们的主要目标是了解遗传因素如何与环境相互作用以打破SD平衡。研究的重点是斑马鱼，这是一种遗传上易受控制的脊椎动物，环境或遗传因素可以将性腺推向卵巢或睾丸的命运。斑马鱼的性腺由双电位器官发育而来，其中一些原始生殖细胞（PGCs）在所有幼鱼中都变成卵母细胞。有些个体卵母细胞死亡，性腺变成睾丸；在其他情况下，卵母细胞存活，性腺变成卵巢。一个关键问题是导致一些幼鱼卵母细胞死亡而另一些幼鱼卵母细胞存活的遗传级联的本质，以及环境因素如何改变这种级联。结果表明，原生SD位点（我们在原生菌株中发现，但从驯化的实验室菌株中消失）与环境因素（可能是可变的营养和生长速率）相互作用，改变幼卵母细胞的存活，进而改变作用于性腺细胞的卵母细胞来源信号的强度，以维持雌激素的产生，从而保存或不保存卵母细胞和性腺的命运。目的一是了解本地斑马鱼SD位点的分子遗传基础。目的2是了解SD期间的转录组级联，使用：a)遗传性别的本地菌株，b)缺乏本地SD基因的环境敏感实验室菌株，以及c)缺乏PGCs的鱼来识别PGC衍生因子及其下游靶标。目的3是通过了解相关TGFss信号因子Gdf9、Amh和Gsdf（性腺细胞衍生因子）的作用，了解性腺细胞/生殖细胞互传信号的机制，并通过诱导突变发现女性特异性信号Wnt4和Foxl2如何在性腺发育中起作用，其中许多我们已经掌握。提出的工作创新包括鉴定和独特使用斑马鱼拥有和对本地SD位点进行基因分型；我们在不同人群中普遍适用的识别性别位点的RAD-sex方法；我们提供的独特的突变体和转基因资源；我们的重点是斑马鱼性别决定中的性腺体细胞/生殖细胞相互信号。这项工作的结果和影响是有可能更全面地了解脊椎动物性别平衡机制的分子遗传学性质，这将增加我们对发达国家人类生殖疾病增加的可能机制的认识。

DESCRIPTION (provided by applicant) Normal human gonad development can be adversely affected in utero by unknown genetic and environmental factors leading to testis dysgeneration syndrome and polycystic ovarian syndrome. In mammals, the Y-chromosome gene SRY initiates an incompletely understood genetic network that directs the gonad to become a testis, while competing genes direct ovary development. Non-mammalian vertebrates use similar down-stream networks, but primary sex determination (SD) genes differ. Additionally, in some species, environmental factors such as temperature or nutrition bias SD. Our broad objective is to learn how genetic factors interact with the environment to tip SD balance. Work focuses on zebrafish, a genetically tractable vertebrate in which environmental or genetic factors can nudge gonads to an ovary or testis fate. Zebrafish gonads develop from bipotential organs in which a few primordial germ cells (PGCs) become oocytes in all juveniles. In some individuals oocytes die and gonads become testes; in others oocytes survive and gonads become ovaries. A key question is the nature of the genetic cascade that causes oocytes to die in some juveniles and survive in others, and how environmental factors alter this cascade. Results suggest the hypothesis that the native SD locus (which we discovered in native strains but is gone from domesticated lab strains) interacts with environmental factors (likely variable nutrition and growth rates) to alter juvenile oocyte survival, which in turn changes the strength of an oocyte-derived signal acting on thegonadal soma to maintain estrogen production, thereby preserving or not preserving oocytes and gonad fate. The aims are: Aim 1 is to learn the molecular genetic basis of the native zebrafish SD locus. Aim 2 is to understand the transcriptomic cascade during SD using: a) a native strain genotyped for genetic sex, b) an environmentally sensitive lab strain that lacks the native SD gene, and c) fish lacking PGCs to identify PGC- derived factors and their downstream targets. Aim 3 is to understand mechanisms of gonadal soma/germ cell reciprocal signaling by learning the roles of the related TGFss signalers Gdf9, Amh, and Gsdf (gonadal soma derived factor), and to discover how the female-specifying signals Wnt4 and Foxl2 act in gonadal development by using induced mutations, many of which we have in hand. Innovation of proposed work includes the identification and unique use of zebrafish possessing and genotyped for the native SD locus; our generally applicable RAD-sex method for identifying sex loci in different populations; the unique mutant and transgenic resources that we make available; and our focus on gonadal soma/germ cell reciprocal signaling in zebrafish sex determination. Outcomes and impact of this work are the potential for a fuller understanding of the molecular genetic nature of the vertebrate sex-balance mechanism, which will increase our appreciation of possible mechanisms underlying increases in human reproductive diseases in developed countries.