Cellule staminali nell'ascidia coloniale Botryllus schlosseri: caratterizzazione di nicchie, fattori di staminalità e dinamiche di differenziamento.

Botryllus schlosseri is a cosmopolitan colonial ascidian pertaining to tunicates, the sister group of vertebrates. It can regenerate entire organisms from a pool of circulating cells when all zooids are removed from a colony. Moreover, it possesses asexual reproduction, in which new individuals continuously develop, while old individuals are resorbed. The close relationship with vertebrates, the constant replacement of old individuals by newly formed zooid generations, together with the high regenerative abilities characterizing this species, make this organism a captivating model for the study of stemness that is the focus of my PhD project. During my PhD, I submitted two book chapters. A first one concerns the methodologies used to study regeneration in colonial and solitary ascidians, since the end of the 19th century until nowadays (Methodological approaches for the study of regeneration in ascidians: an historical overview. Vanni V, Ballarin L, Gasparini F, Peronato A, Manni L (2022). Whole-Body Regeneration: Methods and Protocols. Methods in Molecular Biology, Springer). The long history of studies in ascidians highlights how these organisms are easy to maintain in laboratory conditions, manipulate and follow in vivo. Indeed, the methodologies to induce the regeneration processes have not changed so much, as they are simple, low cost, and effective. However, even if many molecular and cellular tools are still missing for studying these organisms, by applying modern techniques, it has been possible, in recent years, to uncover some features characterizing ascidian stem cells. This is the subject of the second book chapter (Current Knowledge on Stem Cells in Ascidians. Vanni V, Anselmi C, Ballarin L, Drago L, Gasparini F, Gordon T, Peronato A, Rosental B, Rosner A, Rinkevich B, Spagnuolo A, Manni L, Voskoboynik A (2022). Advances in aquatic invertebrate stem cell research, MDPI). Moreover, I followed three different lines to better characterize stemness in B. schlosseri, which are included in different articles. In a paper in collaboration with Prof. Carmela Gissi (Università di Bari, IT), currently under revision, we verified a possible common expression pattern between vertebrate embryonic stem cells and adult candidate stem cells of B. schlosseri. We carefully retrieved the orthologous in B. schlosseri Yamanaka Factors (Myc, Pou2, Pou3 and SoxB1), and studied, through in situ hybridization, the expression of these genes during all the phases of the asexual cycle of this colonial ascidian. Our results highlight a conserved expression pattern of myc, pou3 and soxB1 in undifferentiated, developing territories, consistently with the expression pattern observed in other chordate species. Moreover, the genes are expressed in small round cells (candidate stem cells) located in the stem cell niches identified until now in B. schlosseri, suggesting that they may play a role in stemness. In another paper (in preparation), we characterized the stem cell niches identified until now in B. schlosseri, by 3D reconstruction and histology, showing for the first time their cell structure and anatomy. We then investigated the contribution of candidate stem cells to the development of primary and secondary buds, confirming their infiltration in previously hypothesized sites, but also demonstrating that they can contribute to the development of organs and tissues previously not individuated. Finally, a last paper (in preparation in collaboration with Prof. Jordi Solana, Oxford Brookes University, UK), regards the single cell transcriptomics of B. schlosseri: we successfully produced an atlas of 37 clustered cell populations, validated through published datasets and expressing markers for specific tissues. This atlas will be the basis for future analysis on stemness and differentiation in this organism. Altogether, with the results obtained during my PhD, we advanced the knowledge on stem cells of B. schlosseri.