Robert J. Lauzon

A morphological and immunohistochemical study of programmed cell death in Botryllus schlosseri (Tunicata, Ascidiacea)

The blastogenic cycle of the colonial ascidian Botryllus schlosseri concludes in a phase of selective cell and zooid death called takeover. Every week, all asexually derived parental zooids synchronously regress over a 30-h period and are replaced by a new generation. Here we document the sequential ultrastructural changes which accompany cell death during zooid degeneration. The principal mode of visceral cell death during takeover occurred by apoptosis, the majority of cells condensing and fragmenting into multiple membrane-bounded apoptotic bodies. Cytoplasmic organelles (mitochondria, basal bodies, striated rootlets) within apoptotic bodies retained ultrastructural integrity. Dying cells and fragments were then swiftly ingested by specialized blood macrophages or intraepithelial phagocytes and subsequently underwent secondary necrotic lysis. Certain organs (stomach, intestine) displayed a combination of necrotic and apoptotic changes. Lastly, the stomach, which demonstrated some of the earliest regressive changes, exhibited intense cytoplasmic immunostaining with a monoclonal antibody to ubiquitin at the onset of takeover. Affinity-purified rabbit antiserum against sodium dodecyl sulfate-denatured ubiquitin detected a characteristic 8.6-kDa mono-ubiquitin band by Western blot analysis. Collectively, these findings raise the possibility that cell death during takeover is a dynamic process which requires active participation of cells in their own destruction.

16S Mitochondrial Ribosomal RNA Degradation Is Associated with Apoptosis

The use of mitochondrial RNA as an indicator of apoptosis was investigated. Exposure of HA-1 fibroblastic cells to 10 micromol H(2)O(2) per 10(7) cells induced nuclear fragmentation, cell shrinkage, and internucleosomal DNA fragmentation, all characteristics of apoptosis. RNA extracted from control and apoptotic cultures, and analyzed by Northern blot hybridization, revealed a significant increase in the degradation of mitochondrial 16S ribosomal RNA (rRNA) that was associated with apoptosis. Conversely, minimal, if any, degradation of glyceraldehyde-3-phosphate dehydrogenase or actin mRNAs was observed. Similar results were obtained for HA-1 cells treated with the protein kinase inhibitor staurosporine, and for HT-2 T-lymphocytes induced to undergo apoptosis by interleukin-2 withdrawal. In addition, 16S rRNA degradation was an early event that was discernable well before chromatin condensation in hydrogen peroxide-treated HA-1 cells. These observations suggest that degradation of mitochondrial 16S ribosomal RNA is a new marker of mammalian cell apoptosis.

A morphological study of nonrandom senescence in a colonial urochordate.

Botryllus schlosseri is a clonally modular ascidian, in which individuals (zooids) have a finite life span that is intimately associated with a weekly budding process called blastogenesis. Every blastogenic cycle concludes with a synchronized phase of regression called takeover, during which all zooids in a colony die, primarily by apoptosis, and are replaced by a new generation of asexually derived zooids. We have previously documented that, in addition to this cyclical death phase, entire colonies undergo senescence during which all asexually derived individuals in a colony, buds and zooids, die in concert. In addition, when a specific parent colony (genet) is experimentally separated into a number of clonal replicates (ramets), ramets frequently undergo senescence simultaneously, indicating that mortality can manifest itself in nonrandom fashion. Here, we document a morphological portrait of senescence in laboratory-maintained colonies from Monterey Bay, California, that exhibit nonrandom mortality. Nonrandom senescence proceeded according to a series of characteristic changes within the colony over a period of about one week. These changes included systemic constriction and congestion of the vasculature accompanied by massive accumulation of pigment cells in the zooid body wall (mantle), blood vessels, and ampullae; gradual shrinkage of individual zooids; loss of colonial architecture, and ultimately death. At the ultrastructural level, individual cells exhibited changes typical of ischemic cell death, culminating in necrotic cell lysis rather than apoptosis. Collectively, these observations indicate that senescence is accompanied by unique morphological changes that occur systemically, and which are distinct from those occurring during takeover. We discuss our findings in relation to current experimental models of aging and the possible role of a humoral factor in bringing about the onset of senescence.

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.

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. Developmental Dynamics 234:997–1005, 2005.

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.

Evidence for transcriptional modulation but not acid phosphatase expression during programmed cell death in the colonial tunicate Botryllus schlosseri

Botryllus schlosseri is a clonally modular ascidian in which asexually derived adults (zooids) exhibit developmental synchrony. At the conclusion of the blastogenic (asexual) cycle every 5 days at 21°C, all zooids within a colony die simultaneously in 24 hours and are replaced by a new asexual generation of zooids. This cyclical process, called takeover, involves the selective destruction of the zooids visceral tissues which include the pharynx, esophagus, stomach, intestine, endostyle, neural complex and heart, whereas bud tissues and mesenchymal components (muscle and blood cells) remain unaffected. Ultrastructural analysis indicates that the most prevalent form of cell death occurs by apoptosis, although necrotic changes are also observed in several tissues (i.e., stomach and intestine). Blood‐derived macrophages and neighboring cells subsequently engulf visceral tissues, reducing the zooid to the size of a small vesicle. Here, we have tested the possibility that acid phosphatase, a hydrolase whose presence is associated with cell death in several invertebrate systems, could account for some of the regressive changes observed during takeover. Our observations indicate that acid phosphatase (AP) activity was selectively localized in the gut of parent zooids during the growth phase of the cycle, with the stomach exhibiting the most intense histochemical staining on tissue sections. As zooid regression progressed during takeover, stomach AP staining gradually disappeared. Other visceral tissues never became AP‐positive. Therefore, this hydrolase appears to play a minimal role in zooid death. In order to characterize genes whose expression pattern was selectively altered during takeover, we have carried out differential mRNA display analysis. We report on two genes, 790.3 and 790.4, that are down‐ and upregulated, respectively, during this process. Collectively, these findings indicate that the takeover phase of blastogenesis in Botryllus involves modulated gene expression.

Isolation of Biologically Functional RNA During Programmed Death of a Colonial Ascidian

The blastogenic (asexual) cycle of the colonial ascidian Botryllus schlosseri (Tunicata, Ascidiaceae) concludes in a cyclical phase of programmed cell and zooid death called takeover, in which all asexually derived adults die synchronously by apoptosis. The characterization of developmentally regulated genes whose expression patterns are selectively modulated during this process could pave the way to understand how this model organism dies. However, isolation of biologically functional RNA in this and other colonial ascidians with conventional phenol/chloroform-based procedures is hampered by extensive contamination of RNA preparations by pigments. Upon cell lysis, pigments that normally reside within specialized cells in the mantle wall of the adult are released and tightly associate with nucleic acids. Here, we report on the usefulness of a single-step RNA isolation method in which acid guanidinium isothiocyanate is used as an extraction medium, followed by preparative cesium chloride ultracentrifugation. This procedure successfully isolated biologically active, high-purity total RNA (OD260/OD280 = 1.9-2.1) from Botryllus colonies during takeover, as well as other species of colonial ascidians (Diplosoma macdonaldii, Botrylloides diegense) irrespective of pigmentation. Northern blot analysis performed with a 32P-labeled tunicate actin probe detected two polyadenylated transcripts of 1.5 and 1.7 kilobases in length from both growth phase and takeover colonies. Two-dimensional protein gel assays from in vitro translated mRNA preparations further revealed that specific transcripts were up-regulated during takeover, while others were repressed or down-regulated. Growth phase and takeover-specific cDNA libraries were constructed from pooled poly(A)+ RNA with a complexity of 1.0 x 10(7) and 1.2 x 10(7) recombinants respectively per 100 ng of cDNA before amplification.(ABSTRACT TRUNCATED AT 250 WORDS)