Apoptosis and colonial life cycle in Botryllus schlosseri.

Botryllus schlosseri colonies continuously form new zooids by blastogenesis, through the recurrent formation of palleal buds, which progressively grow and mature until an adult is formed. Three blastogenic generations are commonly co-present: adult, filtering zooids, their buds and budlets on buds. At a temperature of 19°C, adult zooids remain active for about one week (mid-cycle stages); then they contract, close their siphons and are gradually resorbed, being replaced by a new generation of adult zooids, represented by buds which reach functional maturity, open their siphons and begin their filtering activity (regression or take-over stage). This stage is characterised by the occurrence of diffuse programmed cell death by apoptosis, as evidenced by TUNEL reaction for chromatin fragmentation and annexin V labelling for detection of exposed phosphatidylserine (PS), which progressively extends in tissues of adult zooids with an organ gradient. Infiltration of circulating phagocytes, which appear engulfed with apoptotic cells, in zooid tissues is observed. As compared to mid-cycle stages, during the take-over, the frequency of circulating phagocytes showing a globular morphology and containing ingested cells or cell debris (macrophage-like cell; MLC) increases during the regression whereas the frequency of hyaline amebocytes (HA), which represent mobile, active phagocytes, precursors of MLC, decreases. In addition, the number of haemocytes showing nuclear condensation significantly increases as well as the frequency of circulating MLC containing TUNEL-positive cells. The frequency of circulating haemocytes expressing the death receptor Fas progressively rises during the colonial life cycle up to a maximum in the stage immediately before the take-over; their number decreases during the take-over, as they are recognised and engulfed by active phagocytes. Similarly, the frequency of circulating cells expressing Fas ligand (FasL) reaches a maximum immediately before the take-over and remains high until the end of the cycle. During the take-over, the frequency of haemocytes expressing the anti-apoptotic protein Bcl-2 significantly decreases with respect to mid-cycle stages; an opposite behaviour is observed with the pro-apoptotic protein Bax. A similar behaviour is observed in the tissues of the alimentary tract of the zooids. The specific activity of caspase-3, responsible for the activation of nuclear endonucleases, of the haemocyte lysates, is significantly higher during the take-over than in mid-cycle. Phagocytes actively recognise senescent cells and ingest them. When living haemocytes were labelled with the fluorescent stain carboxyfluorescein diacetate and matched in vitro with haemocytes from the same colony, but at different stages of the colonial life-cycle, the number of phagocytes ingesting fluorescent cells was significantly higher if unlabelled haemocytes from mid-cycle stages were incubated together with labelled haemocytes from the take-over than in the case of the opposite combination. As regards the “eat-me” signals on effete cells allowing their recognition and clearance by phagocytes, there is a progressive increase in haemocytes recognised by annexin-V from the beginning of the colonial life-cycle to the take-over. PS seems to be involved in the recognition, as the addition of PS inhibits in vitro phagocytosis of apoptotic cells. CD36, a part of the receptorial complex binding thrombospondin, a bridging molecule between phagocyte surface and apoptotic cells, is expressed on phagocytes: the frequency of cells recognised by anti-CD36 antibodies significantly increases during the take-over with respect to mid-cycle and the expression pattern changes from a patchy distribution on the plasma membrane in mid-cycle to a uniform staining of the phagocyte surface during take-over. In addition, anti-CD36 antibody significantly decreases the phagocytosis of effete cells suggesting that the thrombospondin receptor can play a role in apoptotic cell removal by phagocytes in a manner similar to that described in Vertebrates. Data obtained up to now supports the idea that fundamental mechanisms for the induction and recognition of apoptotic cells are well conserved throughout Chordate evolution.