Hovering between death and life: haemocytes and natural apoptosis in the colonial ascidian Botryllus schlosseri.

Cell death by apoptosis is a fundamental biological process required for the correct sculpturing of developing organs and the controlled elimination of unwanted cells in morphogenesis, regeneration, tissue renewal and maturation of the immune system. B. schlosseri, a cosmopolitan colonial ascidian, undergoes a recurrent generation change or take-over, lasting 24-36 h, during which adult zooids are progressively replaced by growing buds; in the meantime, budlets become buds and new budlet generation appears. During take-over, diffuse, natural apoptosis occurs in zooid tissues, which renders this species an interesting model for the study of apoptosis. The clearance of dying cels is assured by circulating phagocytes which are massively recruited and infiltrate zooid tissues to engulf senescent cels. At the generation change, a significant change in the distribution of circulating phagocytes with respect to mid-cycle, i.e., phases of the bastogenetic cycle lying more than one day from the preceding and following take-over (Lauzon et al. Dev Dyn 194 (1992): 71-83; Manni et al., Dev Dyn 236 (2007): 335-352), occurs and the frequency of circulating hyaline amoeboid phagocytes (hyaline amoebocytes), falls form 25-42% to 12-25%; conversely the percenage of giant phagocytes with ingested materials inside their vacuoles (macrophage-like cells) rises from 4-10% to 20-30% (Cima et al., Cell Tissue Res 312 (2003): 369-376; Ballarin and Cima, Eur J Histochem 49 (2005): 255-264). Since neither adult zooid nor growing buds have opened their siphons during the take-over, in this period a colony does not feed and relies only on the recycling of nutrients deriving form efferocytosis. In addition, during the blastogenetic cycle the amount of dying haemocytes with apoptotic features increases abruptly form 2-5% at mid-cycle to about 30% at take-over: they are characterised by DNA fragmentation phosphatidylserine (PS) exposure on the outer plasma membrane layer and activtion of caspase-9 and -3. New young undifferentiated cells, released from unidentified haemopoietic sites, enter the circulation at this stage (Cima et al., Cell Tissue Res 312 (2003): 369-376; Ballarin et al, Cell Tissue Res 331 (2008): 555-564). In the present work, we present new morphological, cytofluorimetrical and biochemical results useful for a better characterisation of Botryllus apoptosis. Our results indicate that both intrinsic and extrinsic pathways, probably connected by the BH3-only protein Bid, are involved in cell death induction and suggest that oxidative stress may represent the key event in triggering the apoptotic cascade