DynaMO

Female fecundity, i.e. the number of spawned eggs at each reproductive cycle, being the direct output of the oogenetic process occurring in the ovary, is a key factor for the management of both wild and farmed fish. Commitment into oogenesis (frequency, rate) and duration of the overall process greatly vary among fish species. They also significantly vary within a given species in response to extrinsic and intrinsic factors. The mechanisms that govern the cellular dynamics of oogenesis remain however largely unknown. The main reason is that investigating the entire process at the level of the entire organ has been methodologically challenging, if not virtually impossible using classical histology technics based on 2D ovarian sections. For this very reason, we lack a comprehensive overview of the process, and more specifically of the dynamic changes occurring among the different classes/sizes (i.e. oogenetic stages) of oocyte throughout oogenesis, despite several decades of extensive study of the endocrine regulations in the fish ovary. Using the medaka (a small aquarium fish with short generation time widely used to study oogenesis) as a model, we reported a set of 20 new ovarian-specific miRNAs and evidenced the key role of one of them (miR-202) in the determinism of reproductive success and more specifically in fecundity. Besides the preliminary data on the critical role played by miR-202, newly identified miRNAs with predominant ovarian expression suggest an important role of miRNAs in the regulation of fish fecundity. The objective of the DynaMO project is to understand not only the overall dynamics of oogenesis, but also to determine the role of these newly identified miRNAs in the regulation of this process. Our ambition is to go beyond a simple list of key regulatory miRNAs, by providing integrated knowledge of the dynamic of oogenesis in fish and of its regulation by miRNAs. To achieve this aim, we will combine descriptive, functional and modeling approaches, using 3D imaging, genome-editing and mathematics. In the first task of the project we will conduct 3D imaging of the entire intact medaka ovary (using wild-type fish), after tissue clearing, an innovative technology, which is available in the laboratory. This will provide a comprehensive view of oogenesis over time, including the number of oocytes of the different class-sizes present in the ovary. In a second task, carried out simultaneously, we will knock-out specific miRNAs, selected from the pool of newly identified miRNAs, to characterize miRNAs involved in the regulation of fish fecundity. This genome-editing task will be carried out using the CRISPR/Cas9 technology available in the laboratory. The most biologically relevant mutants will then be subjected to extensive histological (3D imaging, task 3) and molecular (RNA-seq and miRNA target prediction, task 4) phenotyping. In the last task of the project, a mathematical model will be built using data from task 1, which will then be used to understand how each mutant affects oogenesis dynamics. Finally, the output of mathematical modeling will be analyzed in the light of the molecular phenotyping performed in task 4 on order to further understand the regulation of oogenesis by miRNA.