2409 Apoptotic cell recognition, engulfment and cell corpse digestion are the final stages in the process of apoptosis. This selective elimination pathway of dying cells is carried out by a variety of phagocytic cells, including macrophages. Studies on C. elegans (Hoeppner et al., 2001; Reddien et al., 2001) and mammals (Diez-Roux et al., 1997; Boyle et al., 2001) revealed that in addition to their classic role of corpse elimination, macrophages may also have a role in the induction of apoptosis in normal cells in vivo or, by engulfing cells, they act to ensure that cells triggered to undergo apoptosis will die, rather than recover after exhibiting initial stages of death. This novel exciting role of macrophages as mediators of the apoptotic process has not been yet documented in whole body apoptotic events. The weekly, colony-wide apoptotic process, characteristic to the urochordate Botryllus schlosseri, offers a unique whole-body apoptotic model system to study this process. Botryllus schlosseri is a colonial marine organism; each colony is composed of numerous genetically identical modules (zooids) and is derived from a single, sexually produced tadpole larva, which immediately settles upon release. The colony grows through a highly synchronized and cyclical developmental phenomenon called blastogenesis (Berrill, 1950). Blastogenesis is divided into four major stages, A–D (Mukai and Watanabe, 1976), and lasts about 1 week (at 18–20°C). It is a highly tuned cycle in which: (1) a new set of zooids is established through the development and maturation of 1–4 primary buds per zooid, and (2) the parent set of zooids deteriorates and is morphologically eliminated. During stages A–C, bud tissues are differentiated and internal organs are formed, while four of the zooids remain active. At stage D, lasting 24–36·h, all zooidal tissues in the colony die, mainly by an apoptotic process, and are phagocyted by specialized blood cells, the macrophages (Lauzon et al., 1992, 1993), which, at this stage, increase infrequency among circulating blood hemocytes (Ballarin et The Journal of Experimental Biology 207, 2409-2416 Published by The Company of Biologists 2004 doi:10.1242/jeb.01045 Apoptosis is an important tool for shaping developing organs and for maintaining cellular homeostasis. In the colonial urochordate Botryllus schlosseri, apoptosis is also the hallmark end point in blastogenesis, a cyclical and weekly developmental phenomenon. Then the entire old generation of zooids are eliminated (resorbed) by a process that lasts 24–36·h. Administration of the antioxidant butylated hydroxytoluene (BHT) resulted in resorption being arrested by 1–8 days on average. At high doses (2.5–15.0·mg·BHT·l –1 ) resorption was completed only after removal of BHT. Colonies that were not removed in time, died. In treated colonies, although DNA fragmentation was high, tissues and organs that would normally have died, survived, and the general oxidative levels of lipids were reduced. Blood vessels were widened, containing aggregates of blood cells with a significantly increased proportion of empty macrophage-like cells without inclusion. In colonies rescued from BHT treatment, resorption of zooids started immediately and was completed within a few days. We propose three possible mechanisms as to how BHT may affect macrophage activity: (1) by interrupting signals that further promote apoptosis; (2) through the respiratory burst initiated following a phagocytic stimulus; and (3) by reducing lipid oxidation and changing cell surface markers of target cells. Our results point, for the first time, to the role of phagocytic cells in the coordination of death and clearance signals in blastogenesis. Key words: apoptosis, phagocytosis, macrophage, BHT, antioxidant, tunicate, B. schlosseri. Summary Introduction Macrophage involvement for successful degeneration of apoptotic organs in the colonial urochordate Botryllus schlosseri Ayelet Voskoboynik 1,2 , Baruch Rinkevich 2, * ,† , Anna Weiss 1 , Elizabeth Moiseeva 2 and Abraham Z. Reznick 1, * 1 Department of Anatomy and Cell Biology, The Bruce Rappaport Faculty of Medicine, Technion – Israel Institute of Technology, Haifa, Israel and 2 National Institute of Oceanography, Oceanographic and Limnological Research, Tel-Shikmona, PO Box 8030, Haifa 31080, Israel *Equal contribution to the manuscript † Author for correspondence (e-mail: buki@ocean.org.il) Accepted 21 April 2004