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.     

Vasa expression in a colonial ascidian, Botrylloides violaceus

Evolution of solitary or colonial life histories in tunicates is accompanied by dramatic developmental changes that affect morphology and reproduction. We compared vasa expression in a solitary ascidian and a closely related colonial ascidian, in an effort to uncover developmental mechanisms important during the evolution of these contrasting life histories, including the ability to reproduce by budding. In this study, we explored the origin of germ cells in new buds developing by asexual reproduction in a colonial ascidian, Botrylloides violaceus and compared it to the source of germ cells in a solitary ascidian Boltenia villosa. We studied expression by in situ hybridization of vasa, a DEAD box RNA helicase gene found in germ cells across the metazoans. In B. villosa, bv-vasa mRNA was expressed in putative germ cells and oocytes of adult gonads, and was sequestered into a posterior lineage during embryogenesis. In mature colonies of the ascidian B. violaceus, bot-vasa mRNA was expressed in putative spermatogonia, in oocytes of zooids, and in some circulating cells in the zooids and differentiating buds. We propose that expression of vasa in cells other than gonadal germ cells of zooids in a colonial ascidian may serve as a source of germ-line stem cells in the colony.     

Programmed cell death in the ascidian embryo: modulation by FoxA5 and Manx and roles in the evolution of larval development

Programmed cell death (PCD) has been discounted in the ascidian embryo because the descendants of every embryonic cell appear to be present in the tadpole larva. Here we show that apoptotic PCD is initiated in the epidermis and central nervous system (CNS) but not in the endoderm, mesenchyme, muscle, and notochord cells during embryogenesis in molgulid ascidians. However, the affected cells do not actually die until the beginning of metamorphosis. Although specific patterns of PCD were different in distantly related ascidian species, the results suggest that removal of CNS cells by apoptosis is a urchordate feature predating the origin of the vertebrates. Certain molgulid ascidian species have evolved an anural (tailless) larva in which notochord cells fail to undergo the morphogenetic movements culminating in tail development. These anural species include Molgula occulta, the sister species of the urodele (tailed) species Molgula oculata. We show that PCD in the notochord cell lineage precedes the arrest of tail development in M. occulta and other independently evolved anural species. The notochord cells are rescued from PCD and a tail develops in hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm, implying that apoptosis is controlled zygotically. Antisense inhibition experiments show that zygotic expression of the FoxA5 and Manx genes is required to prevent notochord PCD in urodele species and hybrids with restored tails. The results provide the first indication of PCD in the ascidian embryo and suggest that apoptosis modulated by FoxA5 and Manx is involved in notochord and tail regression during anural development. Differences in PCD that occur between ascidian species suggest that diversity in programming apoptosis may explain differences in larval form.     

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.     

Haemocytes and blastogenetic cycle in the colonial ascidian <Emphasis Type="Italic">Botryllus schlosseri</Emphasis>: a matter of life and death

A recurrent blastogenetic cycle characterizes colonies of the ascidian Botryllus schlosseri. This cycle starts when a new zooid generation opens its siphons and ends with take-over, when adult zooids cease filtering and are progressively resorbed and replaced by a new generation of buds, reaching functional maturity. During the generation change, massive apoptosis occurs in the colony, mainly in the tissues of old zooids. In the present study, we have investigated the behaviour of haemocytes during the colonial blastogenetic cycle, in terms of the occurrence of cell death and the expression of molecules involved in the induction of apoptosis. Our results indicate that, during take-over, caspase-3 activity in haemocyte lysates increases. In addition, about 20%–30% of haemocytes express phosphatidylserine on the outer leaflet of their plasma membrane, show DNA fragmentation and are immunopositive for caspase-3. Senescent cells are quickly ingested by circulating phagocytes that frequently, having once engulfed effete cells, in turn enter apoptosis. Dying cells and corpses are replaced by a new generation of cells that appear in the circulation during the generation change. This research was supported by the Italian M.I.U.R. (PRIN 2006)     

Delayed insemination results in embryo mortality in a brooding ascidian

We explored the effects of temporal variation in sperm availability on fertilization and subsequent larval development in the colonial ascidian Botryllus schlosseri, a brooding hermaphrodite that has a sexual cycle linked to an asexual zooid replacement cycle. We developed a method to quantify the timing of events early in this cycle, and then isolated colonies before the start of the cycle and inseminated them at various times. Colony-wide fertilization levels (assayed by early cleavage) increased from zero to 100% during the period when the siphons of a new generation of zooids were first opening, and remained high for 24 h before slowly declining over the next 48 h. Because embryos are brooded until just before the zooids degenerate at the end of a cycle, delayed fertilization might also affect whether embryos can complete development within the cycle. Consequently, we also determined the effect of delayed insemination on successful embryo development through larval release and metamorphosis. When fertilization was delayed beyond the completion of siphon opening, there was an exponential decline in the percentage of eggs that ultimately produced a metamorphosed larva at the end of the cycle. Thus, even though the majority of oocytes can be fertilized when insemination is delayed for up to 48 h, the resulting embryos cannot complete development before the brooding zooids degenerate.     

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).     

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.     

Telomerase maintained in self-renewing tissues during serial regeneration of the urochordate Botryllus schlosseri

Telomerase is critical for the protection of germ line and stem cell chromosomes from fatal shortening during replication. In most organisms, telomerase activity is suppressed in progressively committed cells and falls to basal rates in terminally differentiated lineages. The colonial ascidian Botryllus schlosseri propagates asexually and sexually, presumably from pools of stem cells that self-renew throughout the 2- to 5-year colony life span. Asexual budding takes place continuously from the parental body wall. When the colony reaches a critical size, sexual reproduction commences with the generation of gonads. Here, we establish the existence of 6-15 kb telomeres on the ends of Botryllus chromosomes. We develop a real-time quantitative PCR telomeric repeat amplification protocol (TRAP) assay that reliably detects 0.2-100 TPG units in cells and tissues. We find highest levels of enzymatic activity in the gonads, developing embryos, and tissues containing the earliest asexual buds. Telomerase activity appears to be suppressed in later buds during organogenesis and falls to basal rates in mature zooids. We postulate that this pattern reflects maximum telomere restoration in somatic stem cells of early buds and suppression of telomerase activity in progenitors and terminally differentiated cells, indicative of an alternate role for stem cells as repeated body regenerators in colonial life histories.     

Many ways to exit? Cell death categories in plants

Programmed cell death (PCD) is an integral part of plant development and defence. It occurs at all stages of the life cycle, from fertilization of the ovule to death of the whole plant. Without it, tall trees would probably not be possible and plants would more easily succumb to invading microorganisms. Here, we have attempted to categorize plant PCD in relation to three established morphological types of metazoan cell death: apoptosis, autophagy and non-lysosomal PCD. We conclude that (i) no examples of plant PCD conform to the apoptotic type, (ii) many examples of PCD during plant development agree with the autophagic type, and (iii) that other examples are apparently neither apoptotic nor autophagic.     

BS-cadherin in the colonial urochordate Botryllus schlosseri: one protein, many functions

Botryllus schlosseri is a colonial urochordate composed of coexisting modules of three asexually derived generations, the zooids and two cohorts of buds, each at disparate developmental stage. Functional zooids are replaced weekly by the older generation of buds through a highly synchronized developmental cycle called blastogenesis (which is, in turn, divided into four major stages, A to D). In this study, we examined the mode of expression of BS-cadherin, a 130-kDa transmembrane protein isolated from this species, during blastogenesis. BS-Cadherin is expressed extensively in internal organs of developing buds, embryos, ampullae and, briefly, in the digestive system of zooids at early blastogenic stage D (in contrast to low mRNA expression at this stage). In vitro trypsin assays on single-cell suspensions prepared from blastogenic stage D zooids, confirmed that BS-cadherin protein is expressed on cell surfaces and is, therefore, functional. BS-Cadherin expression is also upregulated in response to various stress conditions, such as oxidative stress, injury and allorecognition. It plays an important role in colony morphogenesis, because siRNA knockdown during D/A blastogenic transition causes chaotic colonial structures and disrupts oocytes homing onto their bud niches. These results reveal that BS-cadherin protein functions are exerted through a specific spatiotemporal pattern and fluctuating expression levels, in both development/regular homeostasis and in response to various stress conditions.     

Shaping organisms with apoptosis

Programmed cell death is an important process during development that serves to remove superfluous cells and tissues, such as larval organs during metamorphosis, supernumerary cells during nervous system development, muscle patterning and cardiac morphogenesis. Different kinds of cell death have been observed and were originally classified based on distinct morphological features: (1) type I programmed cell death (PCD) or apoptosis is recognized by cell rounding, DNA fragmentation, externalization of phosphatidyl serine, caspase activation and the absence of inflammatory reaction, (2) type II PCD or autophagy is characterized by the presence of large vacuoles and the fact that cells can recover until very late in the process and (3) necrosis is associated with an uncontrolled release of the intracellular content after cell swelling and rupture of the membrane, which commonly induces an inflammatory response. In this review, we will focus exclusively on developmental cell death by apoptosis and its role in tissue remodeling.     

Apoptosis and phosphatidylserine-mediated recognition during the take-over phase of the colonial life-cycle in the ascidian<Emphasis Type="Italic"> Botryllus schlosseri</Emphasis>

Colonies of the ascidian Botryllus schlosseri undergo recurrent generation changes in which adult zooids are gradually resorbed and replaced by new blastogenic generations. During these periods, known as take-over phases, programmed cell death, which, on the basis of morphological analysis is ascribed to apoptosis, occurs widely in zooid tissues. In the present report, we re-investigate cell death during the take-over process. Results confirm the occurrence of diffuse apoptosis, as evidenced by chromatin condensation, positivity to the TUNEL reaction and expression of phosphatidylserine on the outer leaflet of the plasma membrane. Apoptosis also occurs among haemocytes, and senescent blood cells are actively recognised and ingested by circulating professional phagocytes. Both phosphatidylserine and CD36, a component of the thrombospondin receptor, are involved in the recognition of apoptotic haemocytes, which fosters the idea that fundamental recognition mechanisms are well conserved throughout chordate evolution.     

Vascular regeneration and angiogenic-like sprouting mechanism in a compound ascidian is similar to vertebrates

Tunicates are useful models for comparing differing developmental processes such as embryogenesis, asexual reproduction, and regeneration, because they are the closest relatives to vertebrates and are the only chordates to reproduce both sexually and asexually. Among them, the ascidian Botryllus schlosseri displays high regenerative potential of the colonial circulatory system (CCS). The CCS runs in the common tunic, forming an anastomized network of vessels defined by simple epithelia and connected to the open circulatory system of the zooids. During asexual propagation, new vessels form by means of a tubular-sprouting mechanism, resembling that occurring in other metazoans, particularly during vertebrate angiogenesis. We studied the regeneration of experimentally ablated CCS by analyzing the general dynamics of reorganization of vessels and tunic, their ultrastructure, cell proliferation, and the immunohistology of regenerating structures using antibodies against vertebrate angiogenic factors-vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), epidermal growth factor (EGF), and receptors: VEGFR-1, VEGFR-2, and EGFR. Results show that the regenerative process of CCS occurs by a sprouting mechanism, with participation of angiogenic factors. They also show correspondence between the CCS sprouting of B. schlosseri and angiogenic sprouting in vertebrates, during both normal development and regeneration, and support the idea that this morphogenetic mechanism was co-opted during the evolution of various developmental processes in different taxa.     

A new mode of colony multiplication by modified budding in the ascidian Clavelina gemmae n. sp. (Clavelinidae)

Abstract. A new mechanism of asexual multiplication of colonies is described in a colonial ascidian of the genus Clavelina (Aplousobranchiata). The mechanism consisted of the production of star-shaped buds that originate from a basal vessel that bends anteriorly and extends along the dorsal region of the zooids. Once they are well developed, the buds detached easily and were dispersed by water movement. Analysis of the fine structure of the buds revealed that they were a modification of the stolonic budding common in this genus. Time-lapse video recordings showed that released buds required several days to develop, allowing for a potentially significant dispersal range. The buds underwent organogenesis during which the central part gave rise to a new blastozooid with a defined polarity; the arms of the star gave rise to stolons. A new species is defined based on the presence of this type of budding and on other morphological features. The significance of these findings, which adds to the known mechanisms of asexual reproduction in ascidians, is discussed in relation to the biology and distribution of the species.     

Switching of metabolic-rate scaling between allometry and isometry in colonial ascidians

The metabolic rate and its scaling relationship to colony size were studied in the colonial ascidian Botrylloides simodensis. The colonial metabolic rate, measured by the oxygen consumption rate (V(O2) in millilitres of O(2) per hour) and the colony mass (wet weight M(w) in grams) showed the allometric relationship (V(O2) = 0.0412 M(w)(0.799). The power coefficient was statistically not different from 0.75, the value for unitary organisms. The size of the zooids and the tunic volume fraction in a colony were kept constant irrespective of the colonial size. These results, together with the two-dimensional colonial shape, excluded shape factors and colonial composition as possible causes of allometry. Botryllid ascidians show a takeover state in which all the zooids of the parent generation in a colony degenerate and zooids of a new generation develop in unison. The media for connection between zooids such as a common drainage system and connecting vessels to the common vascular system experienced reconstruction. The metabolic rate during the takeover state was halved and was directly proportional to the colonial mass. The scaling thus changed from being allometric to isometric. The alteration in the scaling that was associated with the loss of the connection between the zooids strongly support the hypothesis that the allometry was derived from mutual interaction among the zooids. The applicability of this hypothesis to unitary organisms is discussed.     

Phenotypic plasticity of reproductive effort in a colonial ascidian,Botryllus schlosseri

Phenotypic plasticity is the capability of a genotype to produce different phenotypes in different environments. Previous studies have indicated phenotypic variability in asexual, male, and female reproduction in Botryllus schlosseri, a hermaphroditic, colonial ascidian, but not explicitly tested for genotype by environment interactions that indicate genetic variation in plastic responses. Consequently, clones derived from an estuarine population were deployed at their native site and a warmer, higher productivity site 10 km up-river. Male reproduction was assayed by testis size, female reproduction by the number of eggs produced, and asexual reproduction by colony growth rate. To test for ontogenetic effects, data were collected from two different generations of zooids born in the field. Analyses of variance indicated plasticity in asexual and female reproduction during the first zooid generation and plasticity in all three traits during the third zooid generation. Reaction norms varied significantly among genotypes in direction and magnitude for asexual reproduction at both times, implying that selection on asexual reproduction is weak. Sperm production during the third zooid generation was significantly lower at the nonnative site, but there was no genotype by environment interaction. The reaction norms for female reproduction varied significantly among genotypes in direction and magnitude during the first zooid generation, but only varied in magnitude during the third generation, with egg production being higher in all genotypes at the nonnative site. Comparisons of weighted frequency distributions between sites demonstrated that differences in egg production in the third generation were due to increases in the proportion of reproductive zooids within a colony. The greater emphasis on female reproduction at a site associated with higher food availability and temperature, and the greater emphasis on male reproduction at a colder, food-limited site, supports predictions from sex allocation theory.     

The gene expression profiling in murine cortical cells undergoing programmed cell death (PCD) induced by serum deprivation

PCD (programmed cell death) is important mechanism for development, homeostasis and disease. To analyze the gene expression pattern in brain cells undergoing PCD in response to serum deprivation, we analyzed the cDNA microarray consisting of 2,300 genes and 7 housekeeping genes of cortical cells derived from mouse embryonic brain. Cortical cells were induced apoptosis by serum deprivation for 8 hours. We identified 69 up-regulated genes and 21 down-regulated genes in apoptotic cells. Based on the cDNA microarray data four genes were selected and analyzed by RT-PCR and northern blotting. To characterize the role of UNC-51-like kinase (ULK2) gene in PCD, we investigated cell death effect by ULK2. And we examined expression of several genes that related with PCD. Especially GAPDH was increased by ULK2. Theses findings indicated that ULK2 is involved in apoptosis through p53 pathway.     

Localization of symbiotic cyanobacteria in the colonial ascidian Trididemnum miniatum (Didemnidae, Ascidiacea)

Trididemnum miniatum is a colonial ascidian harboring the photosymbiotic prokaryote Prochloron sp. These bacterial cells are located in the tunic of the host animal. The present study revealed, by ultrastructural analysis, that the Prochloron cells were exclusively distributed and proliferated in the tunic. They were shown to be embedded in the tunic matrix and to have no direct contact with ascidian cells. Some tunic cells of the ascidians, however, did phagocytize and digest the symbiont. Round cell masses were sometimes found in the tunic and appeared to consist of disintegrating cyanobacterial cells. The thoracic epidermis of ascidian zooids was often digitated, and the epidermal cells extended microvilli into the tunic. Since there were no Prochloron cells in the alimentary tract of the ascidian zooids, the photosymbionts would not be considered part of the typical diet of the host ascidians. Thin layer chromatography showed that the symbionts possessed both chlorophyll a and b, while a 16S rRNA gene phylogeny supported the identification of the photosymbiont of T. miniatum as Prochloron sp.