The molecular basis of sex determination in Nothobranchius furzeri

与其他发育过程（如身体轴的规格或眼睛的发育）相比，性别决定是一种非常可塑的现象。性别决定可以由环境或遗传线索或其组合引起。特别是不同的鱼类物种在性别决定方面表现出显着的多样性。这项建议涉及的一个例子，遗传性别决定在短寿命的非洲鳉Nothobranchius furzeri。由于其寿命短，N。furzeri最近成为研究衰老的一个模型。我们以前的工作表明，N。furzeri使用XX/XY性别决定系统。初步数据表明，在该物种中，Gdf 6 Y是TGF-β家族的成员，起着雄性决定因子的作用。Gdf 6 Y的失活导致完全的雄性到雌性的性逆转，表明Gdf 6 Y是男性性别决定所必需的。然而，我们还没有解决这个问题，它是否也足以诱导雄性途径。此外，我们不明白Gdf 6 Y在性别决定中的功能的基础是获得新的功能还是其表达结构域或表达水平的改变。重要的是，我们还不知道Gdf 6 Y作为TGF-β信号通路的成员在哪一点上进入调节性别决定的分子回路。首先，我们需要获得有关gdf 6 Y及其X染色体编码的对应物gdf 6的时空表达模式的详细信息。这将通过RT-PCR和原位杂交进行。其次，我们将使用不同的启动子过表达gdf 6 Y，并询问这是否足以将性别从雌性逆转为雄性。第三，我们将解决，是否gdf 6 Y和gdf 6的不同mRNA水平的孵化时间点周围的gdf 6 Y mRNA中缺乏microRNA结合位点可能造成的。第四，作为该提案的中心点，我们希望通过使用RNA-Seq的转录组学分析来鉴定Gdf 6 Y的靶标，从而阐明TGF-β信号传导如何影响性别决定。第五，我们想解决gdf 6的作用在进化中是否保守的问题。我们的初步数据表明，在N. furzeri导致眼睛和骨骼畸形。已知具有GDF 6突变的人类患者具有类似的异常。通过灭活N. furzeri，我们想建立一个动物模型，人类疾病与GDF 6失活。总之，通过结合遗传学和分子生物学方法，我们试图深入了解Gdf 6 Y如何启动男性性别决定程序，以及TGF-β信号通路如何整合到调节性别决定基本过程的遗传网络中。

In contrast to other developmental processes like specification of the body axes or eye development, sex determination is an extremely plastic phenomenon. Sex determination can result from environmental or genetic cues, or a combination thereof. Different fish species in particular show a remarkable diversity in how sex determination works. This proposal deals with an example of genetic sex determination in the short-lived African killifish Nothobranchius furzeri. Because of its short lifespan N. furzeri has recently become a model for research on aging. Our previous work has shown that N. furzeri uses an XX/XY sex determination system. Preliminary data suggest that in this species Gdf6Y, a member of the Tgf-β family acts as a male-determining factor. Inactivation of gdf6Y leads to complete male-to-female sex reversal, demonstrating that Gdf6Y is necessary for male sex determination. However, we have not yet addressed the question whether it is also sufficient to induce the male pathway. Also, we do not understand whether the basis for Gdf6Y’s function in sex determination is the acquisition of a novel function or an alteration in its expression domain or expression level. Importantly, we do not yet understand at which point Gdf6Y as a member of the Tgf-β signaling pathway feeds into the molecular circuits that regulate sex determination. At first we need to get detailed information about the temporal and spatial expression pattern of gdf6Y and its X-chromosomally encoded counterpart gdf6. This will be done by RT-PCR and in situ hybridization. Second we will overexpress gdf6Y using different promoters and ask whether this is sufficient to reverse the sex from females to males. Third we will address, whether the different mRNA levels of gdf6Y and gdf6 around the time point of hatching might be caused by the lack of a microRNA binding site in the gdf6Y mRNA. Fourth as a central point of the proposal we want to identify the targets of Gdf6Y by transcriptomic analyses using RNA-Seq and thus shed light on how Tgf-β signaling influences sex determination. Fifth we want to address the question whether the role of gdf6 is conserved in evolution. Our preliminary data suggest that lack of gdf6 function in N. furzeri leads to eye and skeletal malformations. Similar anomalies are known from human patients with GDF6 mutations. By inactivating gdf6 in N. furzeri we want to establish an animal model for human diseases associated with GDF6 inactivation. In summary, by combining genetic and molecular biology approaches we seek to get insights into how Gdf6Y initiates the male sex determination program and how the Tgf-β signaling pathway is integrated in the genetic network regulating the fundamental process of sex determination.