The molecular basis of sex determination in Nothobranchius furzeri

与其他发育过程，如指定身体轴线或眼睛发育不同，性别决定是一种极其可塑性的现象。性别决定可能来自环境或遗传线索，或两者的组合。特别是不同种类的鱼，在性别决定过程中表现出显著的多样性。这项提案涉及了一个关于短命非洲剑鱼Nothobranchius Furzeri的基因性别决定的例子。由于它的寿命很短，最近已经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，在这个物种中，转化生长因子-β家族的一个成员GDF6Y是男性决定因素。Gdf6Y的失活导致完全的男性到女性的性别逆转，表明Gdf6Y是男性性别决定所必需的。然而，我们还没有解决是否也足以诱导男性途径的问题。此外，我们也不知道Gdf6Y在性别决定中的功能基础是获得了一个新的功能，还是其表达结构域或表达水平的变化。重要的是，我们还不知道Gdf6Y作为转化生长因子-β信号通路的成员在什么时候进入了调节性别决定的分子回路。首先，我们需要获得gdf6Y及其X染色体编码的同源基因gdf6的时空表达模式的详细信息。这将通过RT-PCR和原位杂交来完成。其次，我们将使用不同的启动子过表达gdf6Y，并询问这是否足以将性别从雌性逆转为雄性。第三，我们将讨论，在孵化时间点附近，gdf6Y和gdf6的不同mRNA水平是否可能是由于gdf6Y mRNA中缺少microRNA结合位点所致。第四，作为该提案的中心点，我们希望通过使用RNASeq的转录分析来确定Gdf6Y的靶标，从而阐明转化生长因子-β信号如何影响性别决定。第五，我们想要解决的问题是，gdf6在进化中的作用是否保守。我们的初步数据表明，在N.Furzeri中缺乏gdf6功能会导致眼睛和骨骼畸形。在携带GDF6突变的人类患者中也发现了类似的异常。通过在N.Furzeri中灭活gdf6，我们想要建立一个与gdf6失活相关的人类疾病的动物模型。总之，通过结合遗传和分子生物学的方法，我们试图深入了解Gdf6Y如何启动男性性别决定程序，以及转化生长因子-β信号通路如何整合到调节性别决定的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.