UNDIFFERENTIATING CELLS IN THE BLOOD OF THE COLONIAL ASCIDIAN BOTRYLLUS SCHLOSSERI: A MORPHO-FUNCTIONAL CHARACTERISATION.

Colonies of the ascidian Botryllus schlosseri undergo a periodic tissue renewal in the take-over stage of the colonial blastogenetic cycle, during which an extensive apoptosis occurs in the adult zooid tissues and the senescent cells are progressively removed by circulating phagocytes. The haemocytes which circulate in the common vascular system also die partly by apoptosis during this stage. These cells are replaced by new haemocytes, likely differentiating from stem cells. Up to now, haemopoiesis was observed only in solitary ascidians in which haematopoietic noduli were described in the branchial wall. Nothing is known on haemopoiesis in colonial species, in the blood circulation of which two cell types with the morphology of undifferentiated cells are recognizable: haemoblast and lymphocyte. We have studied the cytochemical and immunocytochemical properties of these haemocytes: results indicate the haemoblast as a pluripotent stem cell since it shows a basophilic nucleus labeled either with Hoechst 33342 for euchromatin or anti-Ki-67 and anti-PCNA antibodies specific markers of nuclear proteins involved in cell proliferation and its plasma membrane is labeled by anti-CD34 and anti-CD100 antibodies, specific for haemopoietic cells in vertebrates. Commercial antibodies for cytokine receptors, like interleukin 1 receptor I (IL-1RI) and stem cell factor receptor (SCF-R) label haemoblast plasma membrane, suggesting the presence of growth factor receptors. Both lymphocytes and haemoblasts during the colonial cycle show a significant increase in concentration during the blastogenetic replacement. However, mitosis figures were rarely observed in circulating haemocytes. In vitro assays of haemocyte exposure to colchicine showed the presence of mitosis figures, which significantly increase after exposure to bacteria indicating a proliferating capability in blood circulation mainly as an immune response as observed in other invertebrates like molluscs.