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In Botryllus schlosseri, a highly polymorphic allorecognition system limits the potential for vascular fusion by genetically dissimilar, adjacent colonies. In this issue, Nyholm et al. (2006) uncover the nature of the receptor that recognizes the products of the histocompatibility genes.
Phagocytes of the compound ascidian Botryllus schlosseri are capable of constitutive macropinocytosis (MP) at sites of membrane ruffling along the leading edge. This gives rise to the formation of initially irregular vesicles which then move to the inside of the cells and acquire a more regular morphology. Both phagocyte spreading and MP are enhanced by the recognition of molecules containing the sequence Arg-Gly-Asp (RGD): this suggests that, as in mammals, integrin activation is involved in the induction of both cell spreading and endocytosis. The occurrence of MP is associated with increased oxygen consumption and a rise in the production of superoxide anion, as indicated by nitroblue tetrazolium reduction, and ATP, as indicated by increased cytochrome oxidase activity. On the whole, our results indicate the conservation of common mechanisms of MP induction throughout the Chordate phylum.
Two commonly accepted metabolic theories of aging interpret senescence either in terms of the rate of living, where a fixed total metabolic potential is consumed over an expected lifetime (after which the organism wears out and dies) or, in terms of accumulative oxidative damage resulting in progressive and irreversible changes in metabolic pathways. Protocols based on restricted diets, chronically administered anti-oxidants and the use of established lines of organisms resistant to free radical damage support the metabolic theories of aging by revealing, in many cases, significant extensions of life spans or dramatic anti-aging effects. To test the universality of these metabolic hypotheses of aging, we acutely treated ramets (clonal replicates) from old, long-lived colonies of the urochordate Botryllus schlosseri with lethal doses of the anti-oxidant butylated hydroxytoluene (BHT). This group of organisms has a weekly cyclical and highly synchronized developmental process (blastogenesis), during which all existing zooids are removed by massive apoptosis and phagocytosis processes. In animals treated with BHT, blastogenesis was completely arrested and colonies deteriorated to a morphologically chaotic state. Rescued ramets resorbed BHT treated zooids, regenerated entirely new sets of zooids and then revealed: (1) rejuvenescence and enhanced growth rates and in many cases, (2) up to 4.6 times extension of post-treatment life expectancy. Both metabolic theories for senescence were therefore falsified in B. schlosseri. The possible existence of an aging clock that can be set by the environment is suggested.
The human EBI protein has been cloned by virtue of its interaction with the C-terminus of the APC (adenomatous polyposis coli) protein, whose C-terminal truncated forms have been shown to accompany sporadic and familial forms of colorectal cancer. We have cloned a putative EBI homolog from Botryllus schlosseri (Urochordata. Ascidiacea). The deduced protein is 287 amino acids long, and is identical with 48% of the residues in human EBI and 24-25% in two yeast hypothetical proteins. We propose that such a high degree of conservation among EBI homologs is indicative of an essential regulatory mechanism in eukaryotic cells.
We have initiated a project aimed at documenting molecular and cellular changes underlying the emergence of the hypothalamo-hypophyseal axis in Chordates. Considering the phylogenetic position of Tunicates and the 'pan-hypophyseal' expression pattern of Pitx genes in Vertebrate pituitary, we searched for a Pitx-related homeobox gene in the ascidian Ciona intestinalis, and identified Ci-Pitx (ona intestinalis uitary homeobo gene). We also isolated Cs-Pitx and Bs-Pitx, the Ci-Pitx respective counterparts of Ciona savignyi and Botryllus schlosseri, two other Tunicate species. Ci-Pitx mRNA encodes a putative protein exhibiting the diagnostic K50-Paired-class homeodomain and a conserved C-terminal Aristaless domain. Embryonic expression pattern of Ci-Pitx revealed a conserved expression domain in the anterior neural ridge and subsequently in the pharyngeal primordium, defined in Vertebrates as the stomodeal ectomere, which encompasses the presumptive pituitary territory. This shows that expression at early steps of pituitary development is a feature of Pitx-related genes that was already present in the last common ancestor of Chordates.
The comet assay, one of the most widely used techniques for the evaluation and detection of DNA strand breaks, is frequently employed in vivo. In vitro assays are usually performed with mammalian cell lines, clearly not the best choice for tests on aquatic genotoxicity. Here we evaluated a fish hepatoma cell line (RTH-149) and a primary blood cell culture from the intertidal colonial tunicate Botryllus schlosseri as possible model targets for comet assays using the genotoxic agent H2O2. We found that DNA strand break levels in RTH-149 fitted dose-dependent responses better than the tunicate cells. Moreover, in B. schlosseri controls, 34% of the cells were already ranked as severely damaged. Assays were then performed on water samples from the polluted Kishon river (Israel) on three different dates, using RTH-149 cells (50% dilutions, 2-h exposures). In all cases, high genotoxicity of the river water was revealed by evaluating comet percentages, average tail lengths and DNA damage levels. This assay was found to be fast and sensitive, appropriate to be employed as a part of a monitoring program. The use of B. schlosseri blood cells should be validated in additional work.
Histocompatibility--the ability of an organism to distinguish its own cells and tissue from those of another--is a universal phenomenon in the Metazoa. In vertebrates, histocompatibility is a function of the immune system controlled by a highly polymorphic major histocompatibility complex (MHC), which encodes proteins that target foreign molecules for immune cell recognition. The association of the MHC and immune function suggests an evolutionary relationship between metazoan histocompatibility and the origins of vertebrate immunity. However, the MHC of vertebrates is the only functionally characterized histocompatibility system; the mechanisms underlying this process in non-vertebrates are unknown. A primitive chordate, the ascidian Botryllus schlosseri, also undergoes a histocompatibility reaction controlled by a highly polymorphic locus. Here we describe the isolation of a candidate gene encoding an immunoglobulin superfamily member that, by itself, predicts the outcome of histocompatibility reactions. This is the first non-vertebrate histocompatibility gene described, and may provide insights into the evolution of vertebrate adaptive immunity.
Transplantation immunity based on the recognition of MHC molecules is well described in vertebrates. Vertebrates, however, do not undergo transplantation reaction naturally. The phylogenetically closest group in which transplantation reactions can occur is the Urochordata. Therefore, these animals occupy a key position for understanding the evolution of the vertebrate immune system. When screening for genes differentially expressed during allorecognition in Botryllus schlosseri, we isolated a gene coding for a type II transmembrane protein with a C-type lectin-binding domain and close similarity to vertebrates CD94 and NKR-P1. Here we show that the gene, BsCD94-1, is differentially regulated during allorecognition and that a subpopulation of blood cells carries the corresponding receptor on its cell surface. Southern blot analysis with DNA from individual colonies and intronless BsCD94-1 probe reveal variation between individuals at the genomic level. CD94 in vertebrates is one of the markers for natural killer cells and binds to MHC class I molecules. Natural killer cells play a major role in recognition and elimination of allogeneic cells. Their evolutionary origin, however, remained unknown. The results presented here indicate that the elaboration of the vertebrate immune system may have its roots in an ancestral population of cells in the urochordate blood.
The variety of theories that have attempted to define the mechanisms of aging and life span can be broadly divided into two alternative but nonexclusive viewpoints. The first stipulates that random changes of cellular and molecular structures lead to death following progressive "wear and tear." The second argues that life span is, at least in part, genetically programmed, and therefore aging may also result from time-dependent intrinsic processes. Here we demonstrate that ramets (clonal replicates) experimentally separated from colonies of the acidian protochordate Botryllus schlosseri died months after their separation, almost simultaneously with their parent colony and sibling ramets. In addition, in experimentally joined chimeras between ramets of senescent and nonsenescent colonies, elements from different parent colonies displayed parent-colony-specific timing of mortality. Thus, the senescent phenotype was simultaneously expressed both in chimeras and in unfused ramets of the parent colony that was undergoing senescence, whereas control ramets from the other partner survived. These findings provide experimental evidence for a heritable basis underlying mortality in protochordates, unlinked to reproductive effort and other life history traits of this species.
A colonial protochordate, Botryllus schlosseri, undergoes a natural transplantation reaction in the wild that results alternatively in colony fusion (chimera formation) or inflammatory rejection. A single, highly polymorphic histocompatibility locus (called Fu/HC) is responsible for rejection versus fusion. Gonads are seeded and gametogenesis can occur in colonies well after fusion, and involves circulating germ-line progenitors. Buss proposed that colonial organisms might develop self/non-self histocompatibility systems to limit the possibility of interindividual germ cell "parasitism" (GCP) to histocompatible kin [Buss, L. W. (1982) Proc. Natl. Acad. Sci. USA 79, 5337-5341 and Buss, L. W. (1987) The Evolution of Individuality (Princeton Univ. Press, Princeton]. Here we demonstrate in laboratory and field experiments that both somatic cell and (more importantly) germ-line parasitism are a common occurrence in fused chimeras. These experiments support the tenet in Buss's hypothesis that germ cell and somatic cell parasitism can occur in fused chimeras and that a somatic appearance may mask the winner of a gametic war. They also provide an interesting challenge to develop formulas that describe the inheritance of competing germ lines rather than competing individuals. The fact that fused B. schlosseri have higher rates of GCP than unfused colonies additionally provides a rational explanation for the generation and maintenance of a high degree of Fu/HC polymorphism, largely limiting GCP to sibling offspring.
Theories of evolution that state natural selection acts on individuals have been modified to include multiple levels of selection. Here we demonstrate in chimeric protochordates that primitive germ cell (pgc) and somatic cell (psc) lineages have traits that also make them likely units of natural selection. Specifically, by using microsatellites to determine the genetic identity of various somatic and gametic tissues within vascularly fused Botryllus schlosseri chimeras, we show that genetically distinct pgc and psc can compete for access to developing gonads and somatic organs, and that this competition is hierarchical, reproducible, and heritable. Given that a single, highly polymorphic locus (Fu/HC) controls whether two contacting colonies fuse or reject, our findings also support a leading hypothesis for why the highly polymorphic histocompatibility loci common to many metazoa may have arisen or been maintained: to limit supercompetitor lineages to histocompatible kin.
Germline stem cells (GSCs), the source of gametes, are the only stem cells capable of passing genes to future generations and are therefore considered units of natural selection. Yet, the factors that influence GSC fitness, and thus govern GSC competition, which exist in both protochordates and mammals, remain poorly understood. We studied how aging affects GSC fitness in the protochordate Botryllus schlosseri, an evolutionary crosspoint between invertebrates and vertebrates. GSCs were isolated and distinguished from developing and mature gametes using flow cytometry and scRNA-Seq, facilitated by a new PacBio genome assembly. Moreover, their function was validated through a novel lineage tracing approach that combines membrane-labeled GSC transplantation with scRNA-Seq. Leveraging our method to isolate them, single-cell transcriptomics showed significant age-related changes between young and old GSCs. Spermatids and sperm, however, showed minimal changes, suggesting that reproductive aging is governed by GSCs rather than by gametes. Reduced expressions of markers like DDX4 and PIWIL1 in aged GSCs mirrored trends in mammalian datasets, pointing to a conserved GSC-driven aging mechanism across chordate evolution. This study provides new techniques that lay the foundation to investigate further drivers of GSC fitness and highlights fertility-related genes as promising targets for therapies to preserve reproductive health.
The MHC molecules have been historically perceived as transplantation antigens, though it is now recognized that their primary, if not sole, role is in eliminating parasites and in surveillance and clearance of aberrant self. Indeed, pregnancy in mammals would represent the closest to a natural transplantation process that occurs in vertebrates. However, among the immediate ancestors to the vertebrates, natural intraspecific allorecognition processes are common. Among members of the colonial tunicate Botryllus schlosseri, two individuals that share a single allele of the highly polymorphic fusibility/histocompatibility (Fu/HC) locus are able to fuse with one another. Could this Fu/HC be related to the MHC such that the MHC really did have its origins as a transplantation antigen? Presently we review the genetics and biology of natural transplantation processes in colonial tunicates, comparing it with allorecognition as mediated through the vertebrate T-cell receptor, killer cell inhibitory receptor/Ly49, and MHC. Experimental approaches to determining if the molecules regulating allorecognition in tunicates have any ancestral relationship to the vertebrate MHC are discussed, as is a genomic approach to isolating novel mediators of allorecognition. We also explore the biological basis for allorecognition in colonial tunicates and recent work that highlights the costs of not maintaining a system for allorecognition.
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.
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.
Many benthic marine invertebrates reproduce by releasing sperm into the sea (free-spawning), but the amount of time that sperm are viable after spawning may have different consequences for fertilization, depending on the type of free-spawner. In egg-broadcasting marine organisms, gamete age is usually assumed to be irrelevant because of the low probability of contact between dilute sperm and egg. However, direct dilution effects might be reduced in egg-brooding free-spawners that filter dilute sperm out of the water column, and sperm longevity may play a role in facilitating fertilization in these taxa. We investigated the effects of time, temperature, and mixing on the viability of naturally released sperm of the colonial ascidian Botryllus schlosseri. Our data indicate that B. schlosseri sperm have a functional life span that is considerably longer than those of the sperm of many other marine invertebrate taxa (half-life of approximately 16 to 26 h), are able to fertilize eggs at extremely low external sperm concentrations (ca. 10(1) sperm ml(-1)), and have a longevity that varies with temperature. It is possible that such prolonged sperm longevity may be achieved by reductions in motility, reactivation of quiescent sperm by chemical cues, or intermittent swimming.
The possibility that free-spawning marine organisms may be subject to fertilization failure at low population density (due to the effects of sperm dilution) has sparked much interest, but these effects have been demonstrated only in a few species that broadcast their eggs. Some egg-brooding species may overcome dilution effects by filtering low concentrations of sperm from seawater and fertilizing eggs throughout an extended period of time. We examined the effects of population density and size on fertilization in Botryllus schlosseri, a hermaphroditic colonial ascidian that free-spawns sperm, but broods eggs. We experimentally manipulated the size and density of mating groups and surveyed fertilization levels in natural populations that varied in density. Fertilization was not affected by variation in population size or density in either the experimental or natural populations. Near the end of the reproductive season, some eggs may have been fertilized too late to complete development, suggesting a temporal form of sperm limitation that has not been considered in other systems. We also detected greater variability in fertilization levels at lower population density. Nevertheless, these results suggest that caution must be used in extrapolating reported density effects on fertilization to all taxa of free-spawners; density effects may be reduced in brooders that have efficient sperm collection mechanisms.