The molecular basis of sex determination in Nothobranchius furzeri

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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.