Allorecognition and chimerism in an invertebrate model organism

The presence of highly specific histocompatibility reactions in colonial marine invertebrates that lack adaptive immune systems (such as the sponges, cnidarians, bryozoans and ascidians) provides a unique opportunity to investigate the evolutionary roots of allorecognition and to explore whether homologous innate recognition systems exist in vertebrates. Conspecific interactions among adult animals in these groups are regulated by highly specific allorecognition systems that restrict somatic fusion to self or close kin. In Hydractinia (Cnidaria:Hydrozoa), fusion/rejection responses are controlled by two linked genetic loci. Alleles at each locus are co-dominantly inherited. Colonies fuse if they share at least one haplotype, reject if they share no haplotypes, and display transitory fusion if they share only one allele in a haplotype—a pattern that echoes natural killer cell responses in mice and humans. Allorecognition in Hydractinia and other marine invertebrates serves as a safeguard against stem cell or germline parasitism thus, limiting chimerism to closely related individuals. These animals fail to become tolerant even if exposed during early development to cells from a histoincompatible individual. Detailed analysis of the structure and function of molecules responsible for allorecognition in basal marine invertebrates could provide clues to the innate mechanisms by which higher animals respond to organ and cell allografts, including embryonic tissues.Key words: allorecognition, chimerism, invertebrate, innate immune system     

Model systems of invertebrate allorecognition

Nearly all colonial marine invertebrates are capable of allorecognition--the ability to distinguish between self and genetically distinct members of the same species. When two or more colonies grow into contact, they either reject each other and compete for the contested space or fuse and form a single, chimeric colony. The specificity of this response is conferred by genetic systems that restrict fusion to self and close kin. Two selective pressures, intraspecific spatial competition between whole colonies and competition between stem cells for access to the germline in fused chimeras, are thought to drive the evolution of extensive polymorphism at invertebrate allorecognition loci. After decades of study, genes controlling allorecognition have been identified in two model systems, the protochordate Botryllus schlosseri and the cnidarian Hydractinia symbiolongicarpus. In both species, allorecognition specificity is determined by highly polymorphic cell-surface molecules, encoded by the fuhc and fester genes in Botryllus, and by the alr1 and alr2 genes in Hydractinia. Here we review allorecognition phenomena in both systems, summarizing recent molecular advances, comparing and contrasting the life history traits that shape the evolution of these distinct allorecognition systems, and highlighting questions that remain open in the field.     

EVOLUTIONARY GENETICS OF ALLORECOGNITION IN THE COLONIAL HYDROID HYDRACTINIA SYMBIOLONGICARPUS

Many sedentary, clonal marine invertebrates compete intensively with conspecifics for habitable space. Allorecognition systems mediate the nature and outcome of these intraspecific competitive interactions, such that the initiation of agonistic behavior and the potential for intergenotypic fusion depend strongly on the relatedness of the contestants. The dependence of these behaviors on relatedness, along with the extraordinary precision with which self can be discriminated from nonself, suggest that allorecognition systems are highly polymorphic genetically. However, allotypic specificity of this sort could be produced by any number of genetic scenarios, ranging from relatively few loci with abundant allelic variation to numerous loci with relatively few alleles per locus. At this point, virtually nothing is known of the formal genetics of allorecognition in marine invertebrates; consequently, the evolutionary dynamics of such systems remain poorly understood. In this paper, we characterize the formal genetics of allorecognition in the marine hydrozoan Hydractinia symbiolongicarpus. Hydractinia symbiolongicarpus colonizes gastropod shells occupied by hermit crabs. When two or more individuals grow into contact, one of three outcomes ensues: fusion (compatibility), transitory fusion (a temporary state of compatibility), and rejection (incompatibility, often accompanied by the production of agonistic structures termed hyperplastic stolons). Observed patterns of compatibility between unrelated, half-sib pairs, and full-sib pairs show that unrelated and half-sib pairs under laboratory culture have a very low probability of being fusible, whereas full sibs have a roughly 30% rate of fusion in experimental pairings. The genetic simulations indicate that roughly five loci, with 5–7 alleles per locus, confer specificity in this species. In ecological terms, the reproductive ecology of H. symbiolongicarpus should promote the cosettlement of kin, some of which should be full sibs, and some half sibs. Thus, there is potential for kin selection to play a major role in the evolution of the H. symbiolongicarpus allorecognition system. In genetic terms, this system conforms to theoretical predictions for a recognition system selected to distinguish among classes of kin, in addition to self from nonself.     

Allogeneic interactions in Hydractinia: is the transitory chimera beneficial?

The colonial marine hydroid, Hydractinia, exhibits four possible outcomes to allogeneic contacts: passive rejection, aggressive rejection, stable fusion and transitory fusion. In the special case of transitory fusion, Hydractinia colonies undergo tissue fusion, followed by tissue death at the original contact area, and colony separation. This type of rejection is different in several aspects from the rejection process that accompanies incompatible encounters. It has been suggested that in transitory fusion, the colonies gain immediate benefits from fusion, mainly due to size increase, without succumbing to costs associated with fusion (germ line parasitism). We report a long-term observation of repeated fusion and separation cycles in clones featuring transitory fusion that revealed a slow-down of specific growth rates following fusion, and recovery in growth rates following separation. Very rapid transfer of stained material between partners in transitory chimeras provides suggestive evidence that protection against germ line parasitism is far from being guaranteed by separation. Our data cast doubt as to whether the benefits considered for transitory fusion are sustainable and support the already made suggestion that fusion with self, rather than fusion with kin, has been the major selective force governing the evolution of allorecognition in colonial invertebrates.     

The importance of life-history stage and individual variation in the allorecognition system of a marine sponge

In marine invertebrates with complex life cycles, it may often be the case that trade-offs and behaviors differ between adult and larval stages. In this study, I examined the effects of life-history stage on allorecognition system function in the sponge, Haliclona sp. For sedentary marine invertebrates, allorecognition systems allow individuals to distinguish between genetically similar and distinct tissue they may encounter and are thought to reduce costly tissue fusion with individuals other than self or kin. Although it was found that sessile adults fused preferentially with self-tissue and exhibited a functioning allorecognition system, free-swimming larvae fused equally with sibling and non-sibling larvae resulting in swimming chimeras capable of successful metamorphosis, suggesting a stage-activated allorecognition system. In addition, adult sponges differed significantly in the propensity of their larvae to fuse suggesting variation in parental strategies. Analysis of larval swimming behavior indicated that larvae aggregate and are capable of increasing their encounters with other larvae and perhaps their probability of fusing in nature. The pursuit of fusion at this motile stage, along with evidence of a functioning adult allorecognition system, suggests that larvae may not express a recognition system, or that factors other than relatedness such as benefits to larval or adult chimeras, are involved in larval fusion and a stage-activated allorecognition system. In addition, this study demonstrates the presence of variation among individuals in the allorecognition system's ontogeny in the sponge Haliclona sp.     

Self/non-self Discrimination in Basal Metazoa: Genetics of Allorecognition in the Hydroid Hydractinia

Colonial basal metazoans often encounter members of their ownspecies as they grow on hard substrata, with the encounterstypically resulting in either fusion of close relatives or rejectionbetween unrelated colonies. These allorecognition responsesplay a critical role in maintaining the genetic and physiologicalintegrity of the colony. Allorecognition responses in basalmetazoans are controlled by highly variable genetic systems.The molecular nature of such systems, however, remains to bedetermined. Current efforts to identify the genes and moleculescontrolling allorecognition in basal metazoans have followedtwo pathways: identification of molecules differentially expressedin incompatible interactions, and positional or map-based cloningof allorecognition genes. Most studies following the first approachhave been performed with marine demosponges, while those followingthe second approach have centered on the cnidarian of the genusHydractinia. Here, I discuss the latter, focusing primarilyon the genetic control of allorecognition responses.     

Allorecognition Triggers Autophagy and Subsequent Necrosis in the Cnidarian Hydractinia symbiolongicarpus

Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid Hydractinia symbiolongicarpus when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous in vivo digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion in vivo in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.     

EVOLUTION OF ALLORECOGNITION IN BOTRYLLID ASCIDIANS INFERRED FROM A MOLECULAR PHYLOGENY

Despite the functional and phyletic ubiquity of highly polymorphic genetic recognition systems, the evolution and maintenance of these remarkable loci remain an empirical and theoretical puzzle. Many clonal invertebrates use polymorphic genetic recognition systems to discriminate kin from unrelated individuals during behavioral interactions that mediate competition for space. Space competition may have been a selective force promoting the evolution of highly polymorphic recognition systems, or preexisting polymorphic loci may have been coopted for the purpose of mediating space competition. Ascidian species in the family Botryllidae have an allorecognition system in which fusion or rejection between neighboring colonies is controlled by allele-sharing at a single, highly polymorphic locus. The behavioral sequence involved in allorecognition varies in a species-specific fashion with some species requiring extensive intercolony tissue integration prior to the allorecognition response, while other species contact opposing colonies at only a few points on the outer surface before resolving space conflicts. Due to an apparent species-specific continuum of behavioral variation in the degree of intercolony tissue integration required for allorecognition, this system lends itself to a phylogenetic analysis of the evolution of an allorecognition system. We constructed a molecular phylogeny of the botryllids based on 18S rDNA sequence and mapped allorecognition behavioral variation onto the phylogeny. Our phylogeny shows the basal allorecognition condition for the group is the most internal form of the recognition reaction. More derived species show progressively more external allorecognition responses, and in some cases loss of some features of internal function. We suggest that external allorecognition appears to be a secondary function of a polymorphic discriminatory system that was already in place due to other selective pressures such as gamete, pathogen, or developmental cell lineage recognition.     

Expression of putative immune response genes during early ontogeny in the coral Acropora millepora

Background

Corals, like many other marine invertebrates, lack a mature allorecognition system in early life history stages. Indeed, in early ontogeny, when corals acquire and establish associations with various surface microbiota and dinoflagellate endosymbionts, they do not efficiently distinguish between closely and distantly related individuals from the same population. However, very little is known about the molecular components that underpin allorecognition and immunity responses or how they change through early ontogeny in corals.

Methodology/Principal Findings

Patterns in the expression of four putative immune response genes (apextrin, complement C3, and two CELIII type lectin genes) were examined in juvenile colonies of Acropora millepora throughout a six-month post-settlement period using quantitative real-time PCR (qPCR). Expression of a CELIII type lectin gene peaked in the fourth month for most of the coral juveniles sampled and was significantly higher at this time than at any other sampling time during the six months following settlement. The timing of this increase in expression levels of putative immune response genes may be linked to allorecognition maturation which occurs around this time in A.millepora. Alternatively, the increase may represent a response to immune challenges, such as would be involved in the recognition of symbionts (such as Symbiodinium spp. or bacteria) during winnowing processes as symbioses are fine-tuned.

Conclusions/Significance

Our data, although preliminary, are consistent with the hypothesis that lectins may play an important role in the maturation of allorecognition responses in corals. The co-expression of lectins with apextrin during development of coral juveniles also raises the possibility that these proteins, which are components of innate immunity in other invertebrates, may influence the innate immune systems of corals through a common pathway or system. However, further studies investigating the expression of these genes in alloimmune-challenged corals are needed to further clarify emerging evidence of a complex innate immunity system in corals.     

Differential effect of allorecognition loci on phenotype in Hydractinia symbiolongicarpus (Cnidaria: Hydrozoa)

The allorecognition complex of Hydractinia symbiolongicarpus is a chromosomal interval containing two loci, alr1 and alr2, that controls fusion between genetically distinct colonies. Recombination between these two loci has been associated with a heterogeneous class of phenotypes called transitory fusion. A large-scale backcross was performed to generate a population of colonies (N = 106) with recombination breakpoints within the allorecognition complex. Two distinct forms of transitory fusion were correlated with reciprocal recombination products, suggesting that alr1 and alr2 contributed differentially to the allorecognition response. Specifically, type I transitory fusion is associated with rapid and persistent separation of allogeneic tissues, whereas type II transitory fusion generates a patchwork of continuously fusing and separating tissues.     

The hydroid Hydractinia: a versatile, informative cnidarian representative

The Cnidaria represent the most ancient eumetazoan phylum. Members of this group possess typical animal cells and tissues such as sensory cells, nerve cells, muscle cells and epithelia. Due to their unique phylogenetic position, cnidarians have traditionally been used as a reference group in various comparative studies. We propose the colonial marine hydroid, Hydractinia, as a convenient, versatile platform for basic and applied research in developmental biology, reproduction, immunology, environmental studies and more. In addition to being a typical cnidarian representative, Hydractinia offers many practical and theoretical advantages: studies that are feasible in Hydra like regeneration, pattern regulation, and cell renewal from stem cells, can be supplemented by genetic analyses and classical embryology in Hydractinia. Metamorphosis of the planula larva of Hydractinia can be used as a model for cell activation and communication and the presence of a genetically controlled allorecognition system makes it a suitable model for comparative immunology. Most importantly, Hydractinia may be manipulated at most aspects of its (short) life cycle. It has already been the subject of many studies in various disciplines, some of which are discussed in this essay.     

Autoreactive blood cells and programmed cell death in growth and development of protochordates

The tunicate Botryllus is a marine protochordate whose clonal colonies undergo regulated natural transplantations when they come into contact in nature. The outcome of these transplantations (fusion or rejection) is controlled by genes of a highly polymorphic histocompatibility system that resembles in many respects the mammalian major histocompatibility complex (MHC). While fusion or rejection reactions are often completed within 24 hr after transplantation, resorption of one partner of a pair of fused semiallogeneic colonies may occur days to weeks after initial contact. The latter process is similar to the degeneration of old individuals, or zooids, that precedes maturation of each new generation of asexual buds. Here we describe comparisons of in vitro reactions of a) mixtures of cells from allogeneic animals and b) cells taken from animals at the zooid-resorption ("takeover") stage of colony development. In vitro autoreactivity of cells from resorbing colonies may reflect in vivo responses to senescent cells, which in turn may be related to allorecognition events that govern fusion or rejection between colonies.     

Tunic cell populations during fusion events in the colonial ascidian Didemnum vexillum (Tunicata)

We documented changes in the abundance and distribution patterns of tunic cells involved in the allorecognition response of the colonial aplousobranch Didemnum vexillum, whose zooids do not share a common vascular system. A histological examination of the fusion zone of isogeneic (CIAs) and allogeneic (CAAs) fused colony assays revealed that tunic cuticles were rapidly regenerated. The underlying tunic matrix fused readily in all assays and controls. We identified four different types of tunic cells. Phagocytic cells represented the most abundant cell type in allogeneic fusions, followed by morula cells. These cells were more abundant at the immediate fusion junction than at 120 μm or 240 μm from the junction, most likely because they mediate the allorecognition reaction. Elongated filopodial cells also were present, although only at very low abundances, and a layer of bladder cells was located immediately below the cuticle. Our results provide quantitative evidence for the involvement of tunic cells in the allorecognition response of a highly invasive ascidian.     

KIN INTERACTIONS IN A COLONIAL HYDROZOAN (HYDRACTINIA SYMBIOLONGICARPUS): POPULATION STRUCTURE ON A MOBILE LANDSCAPE

Many sessile colonial organisms intensively compete with conspecifics for growing space. This competition can result in either cooperative fusion or aggressive rejection between colonies, and some species have evolved highly polymorphic genetic systems that mediate the outcome of these interactions. Here we demonstrate the potential for interactions among close kin as the basis for the evolutionary maintenance of a genetically polymorphic allorecognition system in the colonial hydroid Hydractinia symbiolongicarpus, which lives on gastropod shells occupied by hermit crabs. Fusion between hydroids in the laboratory is restricted mainly to encounters between full siblings, whereas other encounters result in aggressive rejection. Natural selection acting on the costs or benefits of fusion between colonies could be responsible for the present maintenance of such a highly specific behavioral response, but only if encounters between fusible colonies still occur in contemporary populations. The large size of these hydroid populations and the mobility of the crabs should limit the potential for interactions among closely related hydroids on the same shell. However, RAPD polymorphisms among a large sample of hydroids from a population off the coast of Massachusetts indicate that genetically similar colonies are often found together on the same shell. Some genetic distances between colonies on the same shell were low relative to genetic distances between colonies on different shells or genetic distances between known full siblings from laboratory matings. We conservatively estimate that 2–18% of co-occurring colonies may be full sibling pairs. These observations suggest that encounters between genetically similar hydroids are common, despite the mobile nature of their habitat, and these encounters may provide frequent opportunities for natural selection to influence the evolution of cooperative and agonistic behaviors and their polymorphic genetic basis.     

The ontogeny of allorecognition in a colonial hydroid and the fate of early established chimeras

Colonies of the marine hydroid, Hydractinia, are able to discriminate between their own tissues and those belonging to unrelated conspecifics. We have studied the ontogeny of this allorecognition system by a series of allogeneic transplantations along a developmental gradient, including two-cell-stage embryos, 8 h morulae, planula larvae and metamorphosed polyps. Allograft acceptance of incompatible tissue was observed in all embryonic and larval stages, whereas metamorphosed polyps rejected incompatible transplanted allografts. Most of the chimeras established at the two-cell-stage, although composed of two allogeneic, incompatible entities with mismatching allorecognition loci, developed normally and remained stable through metamorphosis. The results of post metamorphic transplantation assays among the chimeras and the naive ramets, suggested that both incompatible genotypes were still represented in the chimera despite the onset of alloimmune maturation. The naive colonies always rejected each other. Chimeras established from later embryonic and larval stages did not develop into adult chimeric entities, but rather separated immediately post metamorphosis. We thus show that (1) allorecognition in this species matures during metamorphosis and (2) genetically incompatible entities may coexist in one immunologically mature, chimeric soma, provided that they were grafted early enough in ontogeny.     

Digging for innate immunity since Darwin and Metchnikoff

Immune systems are, increasingly, being studied from comparative perspectives. The analysis of the immunedefense systems of invertebrates, such as fruit flies and earthworms, is an important part of this effort. These systems are innate, natural non‐specific, non‐anticipatory and non‐clonal. This is in contrast to the macrophage T and B systems that characterize vertebrate adaptive immunity whose properties can be categorized as adaptive, induced, specific, anticipatory, and clonal. In this review, we will focus on the earthworm system. Earthworms, like other complex invertebrates, possess several leukocyte types and synthesize and secrete a variety of immunoprotective molecules. The system as a whole effects phagocytosis, encapsulation, agglutination, opsonization, clotting and lysis of foreign components. At least two major leukocytes, small coelomocytes, and large coelomocytes mediate lytic reactions against several targets. Destruction of tumor cells in vitro shows that phagocytosis and natural killer cell responses are distinct properties of coelomocytes. A third type, the chlorogogen cell, synthesizes and sheds effector lytic molecules. Among the lytic molecules, three have been identified and sequenced (fetidins, CCF‐1, lysenin) and another has been discovered (eiseniapore), while three other molecules, H₁ H₂ H₃, share agglutinating and lysing functions. In contrast to these, Lumbricin I is the only known molecule of the earthworm system that is antimicrobial but non‐lytic. Altogether the cellular and humoral components of the earthworm system function to distinguish between self and not self, dispose of internal (cancer?), damaged components and external antigens (microbes). The evolutionary context of the earthworm innate immune system is discussed at the end of this article. BioEssays 24:319–333, 2002. ©2002 Wiley Periodicals, Inc.     

Genetic diversity of the allodeterminant alr2 in Hydractinia symbiolongicarpus

Hydractinia symbiolongicarpus, a colonial cnidarian (class Hydrozoa) epibiont on hermit crab shells, is well established as a model for genetic studies of allorecognition. Recently, two linked loci, allorecognition (alr) 1 and alr2, were identified by positional cloning and shown to be major determinants of histocompatibility. Both genes encode putative transmembrane proteins with hypervariable extracellular domains similar to immunoglobulin (Ig)-like domains. We sought to characterize the naturally occurring variation at the alr2 locus and to understand the origins of this molecular diversity. We examined full-length cDNA coding sequences derived from a sample of 21 field-collected colonies, including 18 chosen haphazardly and two laboratory reference strains. Of the 35 alleles recovered from the 18 unbiased samples, 34 encoded unique gene products. We identified two distinct structural classes of alleles that varied over a large central region of the gene but both possessed highly polymorphic extracellular domains I, similar to an Ig-like V-set domain. The discovery of structurally chimeric alleles provided evidence that interallelic recombination may contribute to alr2 variation. Comparisons of the genomic region encompassing alr2 from two field-derived haplotypes and one laboratory reference sequence revealed a history of structural variation at the haplotype level as well. Maintenance of large numbers of equally rare alleles in a natural population is a hallmark of negative frequency-dependent selection and is expected to produce high levels of heterozygosity. The observed alr2 allelic diversity is comparable with that found in immune recognition molecules such as human leukocyte antigens, B cell Igs, or natural killer cell Ig-like receptors.     

Polymorphism in the Immunoglobulin-like Domains of the Receptor Tyrosine Kinase from the Sponge Geodia Cydonium

Sponges [Porifera] are the phylogenetically oldest phylum of the Metazoa. They are provided with both cellular and humoral allorecognition systems. The underlying molecules are not yet known. To study allorecognition in sponges we first determined the frequency of graft rejection in a natural population of the marine sponge Geodia cvdonium. We then determined, for the first time at the molecular level, the degree of sequence polymorphism in segments of one molecule which may be related to sponge allorecognition and host defense: the Ig-like domains from the receptor tyrosine kinase [RTK]. Thirty six pairs of auto- and allografts were assayed, either by parabiotic attachment or insertion of grafts. All of the autografts fused, while only two allografts fused and 34 pairs were incompatibile. Rejection among the parabiotic allografts was characterized by the formation of a collagenous barrier, while the allografts that were inserted into the host underwent destruction. At the molecular level we first cloned to completion the 5′-end of sponge RTK, which displays a Pro-Ser-Thr-rich sequence; this is thought to act as a module of cell adhesion proteins. Then we analyzed RT-PCR products of amplification across the two Ig-like domains of RTK (about 500 bp), from two pairs of fusing sponges and one pair of rejecting sponges. High levels of polymorphism were recorded, including 18 nucleotide-substitution positions and a tri-nucleotide deletion, which translate into 13 polymorphic amino acid positions. Two of the six sponges were scored as heterozygotes. Among 9 informative polymorphic sites that were tested for linkage disequilibrium, 11 pairwise comparisons were found to be significant, implying the possibility of distinguishable alleles in this locus. To the best of our knowledge this is the first report of polymorphism in Ig-like domains of a receptor from invertebrates that may be associated with allorecognition. This data attests also that fusion in sponges is not confined to genetically identical individuals.     

Allorecognition and xenorecognition in reef corals: a decade of interactions

Xenorecognition phenomena in coral reefs are expressed by a striking array of morphological and cytological responses. Corals encountering conspecifics further elicit additional repertoires of effector mechanisms, specific to allogeneic challenges. Both inducible sets of antagonistic machineries of allo- and xenoresponses are highly specific. In many cases, they are predictable, reproducible, and reveal the hallmark of coral tissue capacity to distinguish between self and non-self. This essay summarizes a decade (1992 –2002) of published results on reef coral immune features. While studies on xenorecognition uncovered the existence of established, non-transitive hierarchies and the importance of antibacterial/cytotoxic compounds secreted by corals, allorecognition assays disclosed the presence of specific and complex non-transitive hierarchies dictated by the expression of a variety of effector mechanisms (‘tailored’ against different conspecifics), the existence of gradual maturation of alloresponses (important in the formation of natural chimeras), the debatable issue of allorecognition memory, and the appearance of delayed, second sets of alloresponses. A critical evaluation of historecognition reveals that expressed responses in different coral systems are phenotypically matched with counterpart outcomes recorded in the mammalian immune systems. Histocompatibility in corals, as in vertebrates, relies on recognition elements (not yet disclosed on the molecular level) as well as on effector mechanisms.