Apoptosis and recognition of apoptotic cells in colonial ascidians

Invertebrate Chordata, collectively named Protochordata, are filter-feeding marine animals, including Cephalochordata and Urochordata. The latter are characterized by a planctonic or sedentary life-style and owe their alternative name of Tunicates to the test or tunic, the peculiar covering in which the body is embedded. They are characterised by the presence of a muscular tail provided with a notochord and a hollow dorsal nerve tube in larval stages of ascidians and thaliaceans where it is resorbed at metamorphosis. Conversely, appendicularians maintain a tail also in adult stages. Tunicates include solitary and colonial forms. Ascidians or sea-squirts are sessile, marine Tunicates diffuse throughout the world, mainly in shallow tropical and temperate waters. About 3,000 species have been reported so far. In colonial ascidians, zooids share a common tunic and are frequently interconnected by a common circulation. In this case, zooids can be either partially isolated but connected by stolons – deriving from the body wall and containing connective tissues and, sometimes, blood vessels- or partially or entirely embedded in the common tunic. In recent years, solitary ascidian species (Ciona intestinalis, Ciona savignyi, Halocynthia roretzi) have emerged as model organisms for the study of the molecular control of embryogenesis and differentiation of specific cell lines (Nishida, 2002; Oda-Ishii et al., 2005; Passamanek and Di Gregorio, 2005; Satoh and Levine, 2005) and their genome was partially or fully sequenced (Dehal etal., 2002; Yokobori et al., 2003). Although less investigated at the molecular level, compound ascidians offer the advantage of the presence of asexual reproduction with the possibility to compare, in the same organism and at various levels (morphological, biochemical, molecular), different developmental pathways (embryogenesis and blastogenesis) leading to the same end product: the adult, filter-feeding zooid. From this viewpoint, it is interesting to note that, among Chordates, a high ability of regeneration and asexual reproduction has been maintained only in Tunicates. The colonial ascidian Botryllus schlosseri is emerging as model organism for the study of asexual reproduction, natural apoptosis and clearance of senescent cells (Lauzon et al., 1992, 1993; Cima et al., 1993), allorecognition (Rinkevich, 1992; Ballarin et al., 2002a; Cima et al., 2004), immunobiology (Ballarin et al., 2000, 2001, 2002b; Cima et al., 2006), regeneration (Tiozzo et al., 2005). B. schlosseri colonies form new zooids by blastogenesis, through the formation of palleal buds which progressively grow and mature until an adult is formed. Three blastogenic generations are commonly found: adult, filtering zooids, their buds and budlets on buds. At a temperature of 19 °C, adult zooids remain active for about a week; 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 (Berrill, 1941; Sabbadin, 1955; Burighel and Schiavinato, 1984; Lauzon et al., 1002). This recurrent generation change in the colonial life-cycle is known as regression or take-over and is characterized by the occurrence of diffuse programmed cell death by apoptosis, as evidenced by TUNEL reaction for chromatin fragmentation and annexin-V labeling for detection of exposed phosphatidylserine (PS), which progressively extends in tissues of adult zooids (Cima et al., 2003). A weekly colonial life cycle can be defined, beginning from the opening of the siphons of a new adult generation and the appearance of a new blastogenic generation, and ending with the resorption of the adult zooids during the take-over. Mid-cycle stages are intermediate stages between the beginning and the ending of the colonial life cycle. During take-over, infiltration of circulating phagocytes, which appear engulfed with apoptotic cells, in zooid tissues is observed. In this period, massive phagocytosis occurs which is paralleled by an increase in both the activity of lysosomal enzymes and the concentration of peroxides in blood plasma, indicating an enhanced phagocytosis and the triggering of a respiratory burst. 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 cells; MLC) increases during the regression whereas the frequency of hyaline amoebocytes, which represent mobile, active phagocytes, decreases. In addition, the number of haemocytes showing nuclear condensation significantly increases as well as the frequency of circulating MLC containing TUNEL-positive cells (Cima et al., 1996, 2003). The frequency of circulating haemocytes expressing the death receptor Fas progressively rises during the colonial life cycle up to a maximun in the stage immediately before the take-over; their number decreases during the take-over, as they are recognized and engulfed by active phagocytes. These cells are mainly represented by old phagocytes and cytotoxic (morula)cells (MC). 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: MLC and MC act as scavenger haemocytes able to induce apoptosis in senescent cells expressing Fas. During the regression phase of the colonial life cycle, 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. Concurrently with the death of effete cells, during the take-over there is the release, in the circulation, of a new generation of young, undifferentiated cells, as revealed by their morphology and their positivity to antibodies raised against CD34, a marker of stem cells in Vertebrates. Phagocytes actively recognise senescent cells an digest them. When living haemocytes were labeled with the fluorescent stain carboxyfluorescein diacetate and matched in vitro with haemocytes form 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 form mid-cycle stages were incubated together with labeled haemocytes from the take-over than in the case of the opposite combination (Cima et al., 2003). Non professional phagocytes, mainly epithelial cells, can occasionally ingest senescent cells. As regards the eat-me signals on effete cells allowing their recognition and clearance by circulating and occasional phagocytes, there is a progressive increase in haemocytes recognized by annexin-V from the beginning of the colonial life-cycle to the take-over. PS seem to be involved in the recognition, as the addition of phospho-L-serine, a soluble analogue of PS, inhibits in vitro phagocytosis of apoptotic cells. Oxidised plasma membrane lipids are also to be important in the interaction between phagocytes and senescent cells as in the presence of antioxidants, phagocytes cannot ingest effete cells. CD36, a part of the receptorial complex binding thrombospondin, a bridging molecule between phagocyte surface and apoptotic cells, is expressed on Botryllus phagocytes: the frequency of cells recognized by anti-CD36 antibodies significantly increases during the take-over with respect to mid-cycle and the expression pattern changes from patchy distribution on the plasma membrane in mid-cycle to a uniform staining of the phagocyte surface during take-over. In addition, anti-CD36 antibodies significantly decrease 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 (Cima et al., 2003). On the whole, data obtained up to now support the idea that fundamental mechanisms for the recognition of apoptotic cells are well conserved throughout Chordate evolution.