Cell Tissue Res (2003) 312:369–376 DOI 10.1007/s00441-003-0738-9 REGULAR ARTICLE Francesca Cima · Giuseppe Basso · Loriano Ballarin Apoptosis and phosphatidylserine-mediated recognition during the take-over phase of the colonial life-cycle in the ascidian Botryllus schlosseri Received: 3 December 2002 / Accepted: 15 April 2003 / Published online: 23 May 2003  Springer-Verlag 2003 Abstract Colonies of the ascidian Botryllus schlosseri undergo recurrent generation changes in which adult zooids are gradually resorbed and replaced by new blastogenic generations. During these periods, known as take-over phases, programmed cell death, which, on the basis of morphological analysis is ascribed to apoptosis, occurs widely in zooid tissues. In the present report, we re-investigate cell death during the take-over process. Results confirm the occurrence of diffuse apoptosis, as evidenced by chromatin condensation, positivity to the TUNEL reaction and expression of phosphatidylserine on the outer leaflet of the plasma membrane. Apoptosis also occurs among haemocytes, and senescent blood cells are actively recognised and ingested by circulating profes- sional phagocytes. Both phosphatidylserine and CD36, a component of the thrombospondin receptor, are involved in the recognition of apoptotic haemocytes, which fosters the idea that fundamental recognition mechanisms are well conserved throughout chordate evolution. Keywords Apoptosis · Recognition · Phagocytes · Ascidian, Botryllus schlosseri (Tunicata) Introduction Cell death by apoptosis is a fundamental process in the development and tissue homeostasis of metazoans. It is involved in the shaping of organs during morphogenesis, in tissue and organ involution, and in the natural turnover of many adult tissues (Wyllie 1987; Samali et al. 1996). Cells undergoing apoptosis are characterised by a series of typical morphological alterations, including cytoplas- mic shrinkage, condensation and fragmentation of nuclear chromatin, surface blebbing and formation of apoptotic bodies, whereas external and internal membranes appear preserved during the process and are able to prevent the induction of inflammatory processes (Kerr et al. 1995; Saraste 1999). In mammals, senescent cells are finally engulfed by professional or occasional phagocytes that recognise biochemical changes on their surface, such as the loss of sialic acid residues from glycoconjugates, the presence of a thrombospondin-binding molecule and the appearance of phosphatidylserine in the outer leaflet of the plasma membrane. Recognition involves scavenger receptors on the surface of phagocytes, such as lectin-like, phosphatidylserine and thrombospondin receptors (Savill et al. 1993; Hart et al. 1996; Fadok et al. 2001a). Three blastogenic generations are usually present in colonies of the ascidian Botryllus schlosseri, i.e. adult filtering zooids, their palleal buds and budlets on buds. At a temperature of 19C, adult zooids remain active for about 1 week; they then contract, close their siphons and are gradually resorbed, being replaced by a new gener- ation of adult zooids, represented by grown buds, which reach functional maturity and open their siphons (Berrill 1941; Sabbadin 1955; Burighel and Schiavinato 1984; Lauzon et al. 1992). During this recurrent generation change in the colonial life-cycle, known as regression or take-over, programmed cell death occurs in tissues of zooids along their antero-posterior axis. This is followed by epidermal contraction and massive infiltration of blood phagocytes (Burighel and Schiavinato 1984; Lauzon et al. 1992). Although some evidence of necrosis has been reported for the digestive system, the bulk of cell death must be ascribed to apoptosis. This assumption is consistent with ultrastructural studies on regressing zooid tissues and the observed changes in ubiquitin immunore- activity (Burighel and Schiavinato 1984; Lauzon et al. This work was supported by the Italian MIUR F. Cima · L. Ballarin ( ) ) Department of Biology, University of Padua, Via Ugo Bassi 58/B, 35121 Padua, Italy e-mail: ballarin@civ.bio.unipd.it Tel.: +39-49-8276197 Fax: +39-49-8276199 G. Basso Department of Pediatrics, University of Padua, Via Giustiniani 3, 35121 Padua, Italy