Periodic Reporting for period 1 - JOLI (Using the jellyfish Clytia hemisphaerica to explore the first steps of meiosis by live-imaging.) Reporting period: 2019-05-01 to 2021-04-30 Summary of the context and overall objectives of the project Meiosis is a fundamental biological process, producing viable high quality gametes is essential for the sexual propagation of most eukaryotic species. Basic research in the field of meiosis enables us to understand how gametes are made, and in turn to understand how and why disruptions in this process can lead to possible infertility, aneuploidy, and also developmental defects. Genetic recombination is a source of variation upon which evolutionary forces can act, but additionally meiosis itself is subject to evolutionary forces. While the progression of meiosis and many of the core components are highly conserved, there is considerable variation among species and even sexes within the same species. As with many other fields, current meiosis knowledge focuses predominantly on classical model systems, however by investigating these processes in new and diverse species, we are able to understand both the diversity of meiotic processes and also gain new insight into the genes and mechanisms that regulate these processes. Our research on Clytia hemisphaerica meiosis provides a crucial evolutionary comparative insight into meiosis in a non-bilaterian animal.The overall objective of this project was to provide a framework for investigating meiosis during oogenesis in Clytia by development of specific molecular tools and approaches to establish a spatiotemporal cartography of early meiotic events within the developing gonad. More specifically the aims were 1) To develop and deploy a variety of tools and methods to characterize early meiosis (in-situ hybridization for gene expression analysis and telomere detection, Clytia specific antibodies for key molecular actors of synapsis to allow their tracking by immunohistochemistry, vital dyes for live imaging), 2) To conduct pilot studies to explore possibilities live imaging of the Clytia gonad, and 3) to study of the function of spo11 in Clytia meiosis via the generation of CRISPR mutants. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Over the course of this project we described the development and organization of the female Clytia hemisphaerica gonad. We developed a number of custom antibodies to visualize key structures and proteins, including for the synaptonemal complex (sycp1) an important meiosis-specific structure that facilitates recombination, piwi, and a centromeric histone. We developed and implemented new protocols for staining with each antibody, a process that required considerable trouble-shooting and optimisation. We also optimized protocols for already tested and commercially available tools, like telomere-FISH, for use with Clytia jellyfish and gonads. Using these tools we have achieved a spatial and also temporal description of the Clytia female gonad as it forms during medusa growth, including entry into meiosis, stages of prophase I, and growing and fully grown oocytes.In pioneering functional studies in this system, we used CRISPR-Cas9 knockout methods to generate several male and female jellyfish lines carrying mutations for Spo11, a key actor in the initiation of meiotic recombination whose precise role during cross species remains to be understood. The mutants showed several clear phenotypes: all spo11 mutants show a clear loss of recombination and pairing in fully grown prophase arrested oocytes, while the formation of the synaptonemal complex was fully or partially abolished. We developed antibodies against the rad-51 protein (optimization ongoing) to assess double stranded breaks. Further analysis showed that after fully grown mutant oocytes are triggered to undergo maturation, meiotic divisions are disrupted such that the resulting female gamete is diploid or tetraploid. Unexpectedly, these eggs can be successfully fertilised , and are capable of producing viable offspring. These results have been disseminated via three scientific conferences either online or in person. Additionally, the project has been communicated to the broader public via science days and lectures. Finally, the results of this project will be written up in two scientific papers to be submitted in the next year, as well as two review papers (one published, another submitted). The data and mutant lines generated in this work will be of use for several future projects, now that Clytia is firmly established as an excellent model to investigate early as well as late events of meiosis. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Meiosis is an important and fundamental process, required for the successful production of sperm and eggs. Defects in the meiotic process can lead to chromosomal diseases, miscarriage, and infertility. Understanding the role and function of key meiosis genes is critical for understanding how mutations in these genes could lead to human infertility, miscarriage, and birth defects. Among the classical animal models, including flies, worms, mice and zebrafish, a diversity of molecular and cellular events underlie key meiotic processes. The phylogenetic distance of Clytia from these animal models provides value, as it provides a key evolutionary perspective on meiosis in animals. Through this study, we have established Clytia hemisphaerica as a highly tractable model for the study of early meiosis. We now have in place tools (including species-specific antibodies), optimised protocols, and established mutant lines, for long term research of meiosis in Clytia hemisphaerica. The gonad organization and spo11 mutant phenotype in Clytia show many important similarities with classical model organisms studied so far, but they differ in ways that leverage investigation of the relationship between DSBs and SC formation, as well as the ways in which embryos can handle extra chromosome copies. Sycp1 (magenta) and DNA (cyan) in a wild type female jellyfish gonad.