138 Current Pharmaceutical Design, 2008, 14, 138-147 1381-6128/08 $55.00+.00 © 2008 Bentham Science Publishers Ltd. A Tale of Death and Life: Natural Apoptosis in the Colonial Ascidian Botryllus schlosseri (Urochordata, Ascidiacea) Loriano Ballarin * , Paolo Burighel and Francesca Cima Department of Biology, University of Padova, Padova, Italy Abstract: The colonial ascidian Botryllus schlosseri forms new zooids by blastogenesis, through the formation of palleal buds which progressively grow and mature until adults are formed. At a temperature of 19°C, adult zooids remain active for about one week; then they contract, close their siphons and are gradually resorbed, being replaced by buds which reach functional maturity, open their siphons and begin their filtering activity as adult zooids. This recurrent generation change, known as take-over, is characterised by the occurrence of diffuse programmed cell death by apoptosis. Immediately before the take-over, an increase in the expression of molecules recognised by anti-Bax antibodies and a parallel decrease in the expression of molecules immunopositive to anti-Bcl-2 antibodies were observed in zooid tissues, suggesting a mitochondrion-dependent apoptotic pathway. During the take-over, circulating phagocytes infiltrate the zooid tissues and engulf apoptotic cells; in addition, the frequency of haemocytes showing nuclear condensation and annexin-V labelling sig- nificantly increases. Previous experiments showed the involvement of phosphatidylserine and CD36 in the recognition of effete cell. The resorption of old zooids is closely related to the rejuvenation of the colony occurring at the take-over. The death of adult zooids puts a quantity of material at the colony disposal. This material is represented by senescent cells, which, once ingested and digested by phago- cytes, can be recycled and used to sustain the burden of blastogenesis: this involves a cross-talk between old tissues, phagocytes and de- veloping buds. Therefore, B. schlosseri can be considered a new and promising model organism for the study of natural apoptosis. Key Words: Botryllus, ascidians, apoptosis, blastogenetic cycle, haemocytes. INTRODUCTION Animals belonging to the phylum Chordata (Vertebrates, Cephalochordates and Tunicates) share four main anatomical char- acteristics: (i) the presence of a permanent or temporary notochord in the form of an elastic dorsal rod, which prevents shortening of the body when longitudinal muscles contract; (ii) a hollow dorsal nerve cord, modified to some extent at the front end; (iii) a ventral gut, which anteriorly forms a pharynx provided with gill slits or pharyngeal pouches, and a ventral glandular structure (endostyle/ thyroid gland) able to fix iodine; and (iv) a muscular tail (post-anal part of the body). Tunicates and Cephalochordates, also named Protochordata or Prochordata, represent approximately 6% of the species of the phy- lum: all of them are small, filter-feeding marine animals. Tunicates or urochordates are invertebrate chordates character- ised by the presence of the test, or tunic, the peculiar tissue which embeds the larval and adult body. Although of epidermal origin, it resembles connective tissues in having amoeboid cells interspersed in an amorphous matrix with tunicine fibres, the latter similar to cellulose in composition. In addition, notochord is restricted in the tail of larvae of ascidians and pelagic tunicates, whereas in appen- dicularians it persists in the adult stage. In adults, the pharynx is well developed, in the form of a branchial basket which occupies most of the body volume. The majority of tunicates is represented by ascidians or sea squirts (approximately 3,000 species described so far), solitary or colonial animals, widespread in shallow tropical and temperate waters of the world and characterised by a sessile life-style [1]. Solitary ascidian species (Ciona intestinalis, Ciona savignyi, Halocynthia roretzi) have recently introduced as model organisms for studying the molecular control of embryogenesis and cell differ- entiation [2-8], and their genome has been partially or fully se- quenced [9-10]. Compound ascidians are less studied at the molecu- lar level but have the advantage that various developmental path- ways (embryogenesis, blastogenesis, and regeneration) leading to *Address correspondence to this author at the Department of Biology, Uni- versity of Padova, Via U. Bassi 58/B, 35100 Padova, Italy; Tel: +39 049 8276197; Fax: +39 049 8276199; E-mail: loriano.ballarin@unipd.it the adult, filter-feeding zooids may be compared in the same organ- ism and at various levels (e.g., morphological, biochemical, mo- lecular) [11]. THE COLONY OF BOTRYLLUS SCHLOSSERI Botryllus schlosseri is a cosmopolitan stolidobranch compound ascidian which represents a well-known model organism for the study of a variety of biological problems, such as sexual and asex- ual reproduction [12-16], regeneration [13, 15, 17-20], allorecogni- tion [14, 21-28], immunobiology [29-31]. A colony of Botryllus schlosseri (Fig. 1a) is formed of: i) sev- eral filter-feeding zooids, approximately 1.5 mm in length, origi- nated by blastogenesis, grouped in star-shaped systems, with the oral siphons in the anterior part of each zooid and a central common cloacal siphon connecting the cloacal chamber, into which individ- ual atrial siphons open, with the exterior; ii) buds (primary buds) on zooids able to replace the parental generation; and iii) budlets (sec- ondary buds) on buds which will grow to buds and, finally, to zooids (Fig. 1b). Zooids, buds, and budlets are embedded in a com- mon, gelatinous and transparent tunic, and are connected by a circu- latory system represented by a network of vessels of epidermal origin, branching out from the zooids and buds, crossing the tunic, and joined to a marginal vessel, which runs along the contour of the colony [32]. Sausage-like blind ends, known as ampullae, depart from these vessels toward the tunic surface and store blood cells. Each colony is a clone, arising from the metamorphosis of a tadpole larva (Fig. 1c), deriving from sexual reproduction. Colonies are hermaphroditic and mature gonads are produced on both sides of the blastozooids after several blastogenetic generations [33, 34]. Gonadogenic potential is higher on the left side of zooids [34, 35]. Male sexual maturity is reached before the appearance of genera- tions maturing both testes and ovaries [34]. B. schlosseri is an ovo- viparous ascidian and fertilisation occurs in the atrial chamber; self fertilisation is prevented by the release of sperms 1-2 days after the ovulation. Similarly to other ascidians, mesolecytic oocytes, ap- proximately 300 m in diameter, are surrounded, by test cells and two (inner and outer) layers of follicle cells. At ovulation, occurring in the close proximity of the generation change or take-over, when the new zooids begin to filtrate, eggs are released in the peribran- chial chamber but maintain a connection with the parent through a