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.
