Finding the evolutionary precursor of vertebrate hematopoietic lineage: Functional and molecular characterization of B. schlosseri immune system

We are characterizing the immune system and cell populations of the tunicate model Botryllus schlosseri on functional and molecular levels. The chordate B. schlosseri belongs to a group that is considered the closest living invertebrate relative of vertebrates. Using FACS analysis we can isolate 12 populations of B. schlosseri cells by size, granularity and auto fluorescence. In order to increase the number of isolated cell populations, we used Cytof to scan large numbers of antibodies. Antibodies that differentially bind to B. schlosseri cells were validated by flow cytometry. Serum against the Botryllus Histocompatibility Factor, and lectins and fluorescent reagents activated by enzymatic activity were also used to differentiate live B. schlosseri cell types. Using these markers we isolated 34 cell populations and created RNA libraries for deep sequencing. Additionally, we developed immunological assays for cytotoxicity, phagocytosis and stem cell tracking. Using gene expression analysis we found hematopoietic stem cells and progenitors; in transplantation assays we showed their capability to induce chimerism of pigmentation and differentiation to other cell types, as well as localization to the stem cell niches. We characterized 3 different phagocytic cell types including a previously undescribed myeloid derived cell population. We describe the B. schlosseri cytotoxic cell population that originates as a large granular lymphocyte-like cell type that become morula cells upon allogeneic challenge activation. Altogether, it seems that the common ancestor of tunicates and vertebrates had a true hematopoietic myeloid lineage, while the cytotoxic cells are a convergent evolutionary system.