Dynamics of Oogenesis as analysed by Live Lightsheet Imaging

• Applicants: Professor Dr. Jan Huisken; Dr. Péter Lénárt
• Subject Area: Cell Biology ; Biophysics ; Developmental Biology
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 505673695

The oocyte is a cell of unique importance for all sexual reproducing animals, providing a haploid genome, all the cellular constituents and the ground plan for embryonic development. Tight regulation of cytoplasmic, cytoskeletal and nuclear events during successive steps of oogenesis is thus essential to avoid both infertility and defects in embryo development. Much of this regulation remains understudied, with a significant limiting factor being experimental access to oocytes developing within the female gonad. An exciting route to overcome such limitations is currently being opened by alternative model organisms, notably ones selected from marine environments, offering a huge range of natural diversity. Marine organisms are typically well suited for imaging approaches, however particular constraints relating to their cool, salt-water environment currently restrict the application of advanced imaging technologies. Our interdisciplinary project aims to overcome these constraints by developing an innovative, tailored light sheet imaging system. We will use this to exploit a jellyfish model, Clytia hemisphaerica, that has high but as yet untapped potential for experimental manipulation and live imaging of oogenesis within the completely transparent animal or in autonomously functioning isolated ovaries. Our research plan includes two technical innovation work-packages and two that address oocyte animal- vegetal polarity establishment, a key process that presages the larval body plan: In WP1 we will develop adaptable custom light sheet microscopes with integrated image acquisition and analysis workflows tailored for marine organisms. In WP2 we will build a suite of molecular tools for expression of tagged proteins and mRNAs during Clytia oogenesis, including transgenic lines. In WP3 we will use these tools in live Clytia ovaries to visualise and dissect functionally how the microtubule cytoskeleton establishes animal-vegetal polarity during the growth phase of oogenesis, focussing on the repositioning of the nucleus and localisation of Fz1 mRNA, one of three key Wnt-pathway axis determinants. In WP4 we will address the mechanisms driving segregation of the two other determinant mRNAs, Fz3 and Wnt3, to opposite cortical domains during oocyte meiotic maturation. For WPs 3 and 4 we will image the cytoskeleton, mRNAs and other key cellular components live in 3D with high spatial and temporal resolution through oocyte growth and maturation. We will combine these with molecular perturbations using inhibitors, morpholinos and genetic tools, as well as physical micromanipulations. Our findings will reveal key conserved mechanisms and illuminate their evolutionary history. More widely, this project will make a significant contribution to oogenesis research by establishing Clytia as a powerful model system and provide tailored light sheet microscopes to exploit the potential of marine model organ- isms for mechanistic studies in biological research.

• DFG Programme: Research Grants
• International Connection: France
• Major Instrumentation: Custom built Light Sheet Microscope
• Instrumentation Group: 5040 Spezielle Mikroskope (außer 500-503)
• Partner Organisation: Agence Nationale de la Recherche / The French National Research Agency
• Cooperation Partner: Professorin Dr. Evelyn Houliston