Final Report Summary - MMEMA (Molecular mechanisms for the evolution of multicellularity in animals) The transition from unicellular protists to multicellular animals (metazoans) is one of the most significant leaps in evolution. The project aimed to unravel the genetic and molecular mechanisms that enabled it. By comparing the gene repertoires of multicellular animals with those of the ancestral protists, we will be able to find a genetic invention at the onset of metazoan multicellularity. The target protists of our project MMEMA include the filastereans Capsaspora owczarzaki and Ministeria vibrans, the closest relatives to the metazoan+choanoflagellate clade (MeCh), and the ichthyosporeans Sphaeroforma arctica and Creolimax fragrantissima, the likely closest ancestors to the Filasterea+MeCh clade. The project objective includes analyses of their whole genome sequences, of which all, except C. fragrantissima, are being sequenced by the international project UNICORN, where the coordinator is one of the core members. Our project especially focused on gene families serving multicellular-specific functions in metazoans such as signal transduction, which play major roles in cell-cell communication. Co-option (recruiting pre-existing genes for other roles in different times and places) is also an important mechanism to innovate a new system. To uncover such functional transitions during evolution of multicellularity, we need to compare gene functions before and after the multicellularity evolved. However, there had been no model protist that allows such functional analyses. The project therefore aimed at establishing protocols of molecular- and genetic-level experiments including transformation on our target protists. The performed objectives and their main results are the followings. 1) Hiroshi Suga (HS), the beneficiary researcher, analysed the whole genome sequence of C. owczarzaki, especially focused on diversity of protein tyrosine kinases (TKs), and identified 103 TKs in this protist. He also analysed other novel or already-published genome sequences that are relevant to the evolution of multicellularity. He found that the basic repertoire of cytoplasmic TKs (CTKs) has been established well before the origin of multicellularity, whereas receptor TKs (RTKs) independently diversified in each of metazoan, choanoflagellate, and filasterean clades. We concluded that co-opting RTKs that had been used for detecting environmental cues for other functions such as cell-cell communication, and creating metazoan-specific repertoire specially optimised for such functions may have been one of the key changes at the onset of animal multicellularity (Science Signaling, in review). HS also sequenced the genome of C. fragrantissima, using a part of the project's budget. The obtained genome sequence is particularly important because C. fragrantissima is currently the only unicellular relative of Metazoa in which molecular approaches are possible (see below). 2) Using a cell sorter, HS and his colleague have separated the S. arctica cells with single nucleus from those with multiple nuclei and colonies. The extracted RNA was sent to the Broad Institute (United States) and the deep transcriptome sequencing is being performed. We should be able to identify the genes responsible for colony formation by comparing these two transcriptomes. 3) Lastly but most importantly, HS has succeeded in transforming C. fragrantissima by DNA constructs, which is the first success in transforming the metazoan relative protists. By investigating time-laps movies of live cells expressing histone-GFP and studying the sub-cellular structures by antibody staining, HS has elucidated unique life cycles of ichthyosporeans including strictly synchronised nuclear division in a syncytium (manuscript in preparation). We conclude, though there was a delay in the overall progress, that our project made a significant impact on the growing community of European evo-devo and successfully disseminated the 'seeds' of new research field which are going to bloom in a few years.