Phagocyte dynamics in a highly regenerative urochordate: insights into development and host defense.

Phagocytosis is a cellular process by which particles and foreign bodies are engulfed and degraded by specialized cells. It is functionally involved in nutrient acquisition and represents a fundamental mechanism used to remove pathogens and cellular debris. In the marine invertebrate chordate Botryllus schlosseri, cell corpse engulfment by phagocytic cells is the recurrent mechanism of programmed cell clearance and a critical process for the successful execution of asexual regeneration and colony homeostasis. In the present study, we have utilized a naturally occurring process of vascular parabiosis coupled with intravascular microinjection of fluorescent bioparticles and liposomes as tools to investigate the dynamics of phagocyte behavior in real-time during cyclical body regeneration. Our findings indicate that B. schlosseri harbors two major populations of post-mitotic phagocytes, which display distinct phagocytic specificity and homing patterns: a static population that lines the circulatory system epithelia, and a mobile population that continuously recirculates throughout the colony and exhibits a characteristic homing pattern within mesenchymal niches called ventral islands (VI). We observed that a significant proportion of ventral island phagocytes (VIP) die and are engulfed by other VIP following takeover. Selective impairment of VIP activity curtailed zooid resorption and asexual development. Together, these findings strongly suggest that ventral islands are sites of phagocyte homing and turnover. As botryllid ascidians represent invertebrate chordates capable of whole body regeneration in a non-embryonic scenario, we discuss the pivotal role that phagocytosis plays in homeostasis, tissue renewal and host defense.

Asexual propagation and regeneration in colonial ascidians.

Regeneration is widely distributed among the metazoans. However, clear differences exist as to the degree of regenerative capacity: some phyla can only replace missing body parts, whereas others can generate entirely new individuals. Ascidians are animals that possess a remarkable regenerative plasticity and exhibit a great diversity of mechanisms for asexual propagation and survival. They are marine invertebrate members of the subphylum Tunicata and represent modern-day descendants of the chordate ancestor; in their tadpole stage they exhibit a chordate body plan that is resorbed during metamorphosis. Solitary species grow into an adult that can reach several centimeters in length, whereas colonial species grow by asexual propagation, creating a colony of genetically identical individuals. In this review, we present an overview of the biology of colonial ascidians as a paradigm for study in stem cell and regenerative biology. Focusing on botryllid ascidians, we introduce the potential roles played by multipotent epithelia and multipotent/pluripotent stem cells as source of asexual propagation and regenerative plasticity in the different budding mechanisms, and consider the putative mechanism of body repatterning in a non-embryonic scenario. We also discuss the involvement of intra-colony homeostatic processes in regulating budding potential, and the functional link between allorecognition, chimerism, and regenerative potential.

Suppression of programmed cell death regulates the cyclical degeneration of organs in a colonial urochordate.

The survival of animal tissues and organs is controlled through both activation and suppression of programmed cell death. In the colonial urochordate Botryllus schlosseri, the entire parental generation of zooids in a colony synchronously dies every week as the asexually derived generation of buds reaches functional maturity. This process, called takeover, involves massive programmed cell death (PCD) of zooid organs via apoptosis followed by programmed removal of cell corpses by blood phagocytes within approximately 1 day. We have previously reported that developing buds in conjunction with circulating phagocytes are key effectors of zooid resorption and macromolecular recycling during takeover, and as such engineer the reconstitution of a functional asexual generation every week [Lauzon, R.J., Ishizuka, K.J., Weissman, I.L., 2002. Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration. Dev. Biol. 249, 333-348]. Here, we demonstrate that zooid lifespan during cyclic blastogenesis is regulated by two independent signals: a bud-independent signal that activates zooid PCD and a bud-dependent, survival signal that acts in short-range fashion via the colonial vasculature. As zooids represent a transient, mass-produced commodity during Botryllus asexual development, PCD regulation in this animal via both activation and suppression enables it to remove and recycle its constituent zooids earlier when intra-colony resources are low, while maintaining the functional filter-feeding state when resources are adequate. We propose that this crosstalk mechanism between bud and parent optimizes survival of a B. schlosseri colony with each round of cyclic blastogenesis.

Identification of a novel gene involved in asexual organogenesis in the budding ascidian Botryllus schlosseri.

Development via regeneration or budding shares some known genetic pathways with embryogenesis, but no concerted effort has been made to identify genes unique to asexual development. We have identified a novel gene that plays a role in cyclical bud formation and asexual organogenesis in the colonial ascidian Botryllus schlosseri. Athena mRNA is transcribed at high levels during the 24- to 36-hr interval of programmed cell death and new bud initiation at the conclusion of the budding cycle (takeover). Knockdown of Athena by RNAi and antisense morpholinos induced defects in the development of new buds ranging from retardation in growth and abnormal organogenesis to hollow buds lacking organs. As genetic intervention in this organism has not been possible, this study establishes the use of RNAi and morpholinos in Botryllus as well as describing the knockdown phenotype of a new gene.

Cyclical generation and degeneration of organs in a colonial urochordate involves crosstalk between old and new: a model for development and regeneration.

Botryllus schlosseri is a colonial marine urochordate in which all adult organisms (called zooids) in a colony die synchronously by apoptosis (programmed cell death) in cyclical fashion. During this death phase called takeover, cell corpses within the dying organism are engulfed by circulating phagocytic cells. The "old" zooids and their organs are resorbed within 24-36 h (programmed cell removal). This process coincides temporally with the growth of asexually derived primary buds, that harbor a small number of undifferentiated cells, into mature zooids containing functional organs and tissues with the same body plan as adult zooids from which they budded. Within these colonies, all zooids share a ramifying network of extracorporeal blood vessels embedded in a gelatinous tunic. The underlying mechanisms regulating programmed cell death and programmed cell removal in this organism are unknown. In this study, we extirpated buds or zooids from B. schlosseri colonies in order to investigate the interplay that exists between buds, zooids, and the vascular system during takeover. Our findings indicate that, in the complete absence of buds (budectomy), organs from adult zooids underwent programmed cell death but were markedly impaired in their ability to be resorbed despite engulfment of zooid-derived cell corpses by phagocytes. However, when buds were removed from only half of the flower-shaped systems of zooids in a colony (hemibudectomy), the budectomized zooids were completely resorbed within 36-48 h following onset of programmed cell death. Furthermore, if hemibudectomies were carried out by using small colonies, leaving only a single functional bud, zooids from the old generation were also resorbed, albeit delayed to 48-60 h following onset of programmed cell death. This bud eventually reached functional maturity, but grew significantly larger in size than any control zooid, and exhibited hyperplasia. This finding strongly suggested that components of the dying zooid viscera could be reutilized by the developing buds, possibly as part of a colony-wide recycling mechanism. In order to test this hypothesis, zooids were surgically removed (zooidectomy) at the onset of takeover, and bud growth was quantitatively determined. In these zooidectomized colonies, bud growth was severely curtailed. In most solitary, long-lived animals, organs and tissues are maintained by processes of continual death and removal of aging cells counterbalanced by regeneration with stem and progenitor cells. In the colonial tunicate B. schlosseri, the same kinds of processes ensure the longevity of the colony (an animal) by cycles of death and regeneration of its constituent zooids (also animals).