Mechanism of Female Fate Determination and Maintenance in Zebrafish

斑马鱼作为生物医学研究的重要模式生物已有30多年的历史。令人惊讶的是，斑马鱼如何决定它们将成为哪种性别，目前还不清楚。在人类中，性别是由从父母那里继承的特定染色体决定的，其中XY个体是男性，XX个体是女性。然而，在斑马鱼中还没有发现这种特殊的染色体。包括德雷珀实验室在内的几个实验室最近的研究发现，卵母细胞是卵子的前体，是女性发育和维持女性命运所必需的。不能产生卵母细胞的斑马鱼发育和雄鱼，以及失去卵母细胞的成年雌性鱼恢复到雄鱼。这些研究得出结论，卵母细胞产生一种信号，起到促进和维持女性发育的作用。德雷珀实验室已经确定了两个候选的卵母细胞产生的信号分子，它们拥有强有力的初步数据，表明一个分子的功能是启动女性发育，而另一个分子的功能是维持成年女性的发育。有了这个奖项，他们将进一步确定这些信号如何调节男性和女性发育之间的选择。这些研究的完成将提供有关斑马鱼性别如何确定的基础知识，这对其他从事斑马鱼研究的科学家来说将是重要的，但也将为其他鱼类物种的这一过程提供见解。最后，了解这些信号如何影响斑马鱼的性别，可能有助于确定以前未知的途径，环境污染物可以通过这些途径影响野生鱼类种群的性别比例和功能。这一点非常重要，因为鱼类是世界上许多人口的重要食物来源，由于过度捕捞、污染和栖息地破坏，野生鱼类种群正面临巨大且不断增加的压力。除了这些好处，这项研究还将为代表性不足和/或处于不利地位的研究生、本科生和高中生提供指导和有意义的科学研究经验。斑马鱼是向学生介绍前沿生物学研究的理想模式生物。斑马鱼是世界上数百个实验室使用的主要模式生物。然而，斑马鱼性别决定的机制仍然不清楚。最近的几项研究已经确定，性别决定不是由单一的遗传基因决定的，而产生卵母细胞的能力是女性决定的先决条件。虽然斑马鱼成年后不会改变性别，但Draper实验室最近发现，保持成年雌性表型是一个活跃的过程，需要从卵母细胞到体细胞性腺的持续信号。综上所述，这些研究表明，卵母细胞衍生的信号(S)影响体细胞性腺的命运，而体细胞性腺又通过产生激素来调节动物的显性表型。有几个关键问题仍有待解决：1)卵母细胞衍生信号的性质？2)卵母细胞衍生信号的功能是什么？3)雌性初级决定和成年雌性表型的维持之间是否存在机械联系？工作假设是，卵母细胞信号促进体细胞性腺产生雌激素，雌性初级决定和维持成体表型需要一个阈值水平的雌激素。如果没有这一点，体细胞性腺默认为雄性发育程序，导致幼虫的雄性初级决定，或成年雌性到雄性的逆转。这些预测将通过基因敲除和转基因技术的组合在三个目标中得到验证：特定目标1将测试体细胞性腺(特别是颗粒细胞)产生的雌激素是否必要且足以促进女性初步确定和维持成年女性的表型。具体目标2将确定规范的Wnt信号对于雌性初级决定是否是必要和充分的。具体目标3将测试卵母细胞表达的细胞信号分子Bmp15促进颗粒细胞发育、雌激素产生和维持雌性决定的必要性。预计这些研究将对我们理解2%的硬骨鱼物种成年后恢复的机制产生重大影响。此外，它们应该导致允许对斑马鱼和其他鱼类物种的性别比例进行监管的方法。最后，了解斑马鱼生殖细胞-体细胞性腺信号回路的详细机制可能会确定以前未知的途径，环境污染物可以通过这些途径影响野生鱼类种群的性别比例和功能。

The zebrafish has been an important model organism for biomedical research for over 30 years. Surprisingly, it is still not known how zebrafish determine which gender they will become. In humans, gender is determined by the specific chromosomes that are inherited from the parents, were XY individual are male and XX individuals are female. However, in zebrafish no such specific chromosomes have been identified. Recent studies from several labs, including the Draper lab, have found that oocytes, the precursor to eggs, are required for female development and for maintenance of the female fate. Zebrafish that cannot produce oocytes develop and males, and adult female fish that lose their oocytes revert to males. These studies have concluded that oocytes produce a signal that functions to promote and maintain female development. The Draper lab has identified two candidate oocyte-produced signaling molecules and they have strong preliminary data that indicates that one functions to initiate female development, while the other functions to maintain female development in adults. With this award they will further determine how these signals regulate the choice between male and female development. Completion of these studies will provide fundamental knowledge about how zebrafish gender is determined, which will be important for other scientist working with zebrafish, but will also provide insights into this process in other fish species. Finally, understanding how these signals influence gender in zebrafish may lead to the identification of previously unknown pathways by which environmental pollutants can affect gender ratios and function in wild fish populations. This is of major importance as fish are a vital food source for much of the world's population and wild fish stocks are under intense and increasing pressures that are due to over-fishing, pollution and habitat destruction. In addition to these benefits, this study will also provide mentorship and meaningful scientific research experience to underrepresented and/or disadvantaged graduate, undergraduate and high school students. The zebrafish is an ideal model organism for introducing students to cutting edge biological research.The zebrafish is a major model organism that is used by hundreds of labs around the world. Yet, the mechanism of zebrafish gender determination remains unknown. Several recent studies have determined that no single genetic locus is responsible for gender determination, and that the ability to produce oocytes is a prerequisite for female determination. While zebrafish do not switch gender as adults, the Draper lab has recently discovered that maintenance of the adult female phenotype is an active process that requires continuous signaling from oocytes to the somatic gonad. Together these studies suggest that an oocyte-derived signal(s) influences the fate of the somatic gonad, which in turn regulates the overt phenotype of the animal through production of hormones. Several key questions remain to be resolved: 1) what is the nature of the oocyte-derived signal? 2) What is the function of the oocyte-derived signal? 3) Is there a mechanistic link between female primary determination and maintenance of the adult female phenotype? The working hypothesis is that the oocyte signal promotes estrogen production by the somatic gonad, and that a threshold level of estrogen is required for female primary determination and maintenance of the adult phenotype. Absent this, the somatic gonad defaults to a male developmental program, resulting in male primary determination in larvae, or female-to-male reversal in adults. These predictions will be tested in three aims using a combination of gene knockout and transgenic technologies: Specific Aim 1 will test if estrogen production by the somatic gonad (specifically by granulosa cells) is necessary and sufficient to promote female primary determination and maintenance of the adult female phenotype. Specific Aim 2 will determine whether canonical Wnt signaling is necessary and sufficient for female primary determination. Specific Aim 3 will test the necessity of the oocyte-expressed cell-signaling molecule Bmp15 to promote granulosa cell development, estrogen production, and maintenance of female determination. It is anticipated that these studies will have significant impact on our understanding of the mechanism that allows 2% of teleost species to revert as adults. In addition, they should lead to methods that allow the regulation of gender ratios in zebrafish and perhaps other fish species. Finally, knowing the detailed mechanism of the germ cell-somatic gonad signaling circuitry in zebrafish may identify previously unknown pathways by which environmental pollutants can affect gender ratios and function in wild fish populations.