Phagocyte localization and function during organismal apoptosis and regeneration in botryllus schlosseri

Programmed cell death (PCD) is an important part in the development and maintenance of homeostasis in multicellular animals. We utilized the colonial marine urochordate Botryllus Schlosseri as a model system to investigate this process because it is an organism whose adult generation (zooids) cyclically undergoes massive PCD in all of its organs every 5 days at 21oC. Specialized blood phagocytes have previously been shown to play a major role in the clearance of apoptotic cell corpses during this death phase. Here, using fluorescence stereomicroscopy and laser-scanning confocal microscopy, we investigated phagocyte localization and function during colony homeostasis in Botryllus colonies via intravascular microinjection of fluorescent bioparticles and liposomes containing the phagocidal drug clodronate. Our findings indicate that both bioparticles and liposomes were rapidly targeted and engulfed within phagocytic cells localized within ventral islands of adult zooids. Knockdown of phagocyte function using clodronate liposomes further revealed that phagocytes were important throughout the blastogenic cycle of this animal, but most notably during the PCD phase. Clodronate colonies showed abnormalities consistent with phagocyte disruption. Using co-microinjection of liposomes and bioParticles, phagocyte populations were also observed to engulf foreign particles with different levels of specificity. The stationary endothelial cells only engulfed bioParticles, while migratory blood phagocytes ingested both types of particles. Collectively, our observations indicate that blood phagocytes are critical effectors in colony regeneration and homeostasis. Finally, in order to characterize molecular markers expressed in Botryllus phagocytic cell populations, a 180bp segment of a putative scavenger receptor homolog was also successfully isolated and amplified