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.     

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.     

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.     

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.     

An invertebrate histocompatibility complex

We have developed defined genetic lines of the hydroid Hydractinia symbiolongicarpus and confirmed earlier results showing that allorecognition is controlled by a single chromosomal region within these lines. In a large backcross population, we detected recombinants that display a fusibility phenotype distinct from typical fusion and rejection. We show that this transitory fusion phenotype segregates in a fashion expected of a single Mendelian trait, establishing that the chromosomal interval contains at least two genes that interact to determine fusibility. Using bulked segregant analysis, we have identified amplified fragment length polymorphisms (AFLP) cosegregating with fusibility, used these markers to independently confirm linkage of the two loci, and constructed a 3.4-cM map of an invertebrate histocompatibility complex.     

Experimental demonstration of the benefits of somatic fusion and the consequences for allorecognition

Allorecognition, the ability to distinguish “self” from “nonself” based on allelic differences at allorecognition loci, is common in all domains of life. Allorecognition restricts the opportunities for social parasitism, and is therefore crucial for the evolution of cooperation. However, the maintenance of allorecognition diversity provides a paradox. If allorecognition is costly relative to cooperation, common alleles will be favored. Thus, the cost of allorecognition may reduce the genetic variation upon which allorecognition crucially relies, a prediction now known as “Crozier's paradox.” We establish the relative costs of allorecognition, and their consequences for the short‐term evolution of recognition labels theoretically predicted by Crozier. We use fusion among colonies of the fungus Neurospora crassa, regulated by highly variable allorecognition genes, as an experimental model system. We demonstrate that fusion among colonies is mutually beneficial, relative to absence of fusion upon allorecognition. This benefit is due not only to absence of mutual antagonism, which occurs upon allorecognition, but also to an increase in colony size per se. We then experimentally demonstrate that the benefit of fusion selects against allorecognition diversity, as predicted by Crozier. We discuss what maintains allorecognition diversity.     

Colonial allorecognition,hemolytic rejection,and viviparity in botryllid ascidians

Allorecognition is a fundamental system that animals use to maintain individuality. Although embryos are usually semiallogeneic with their mother, viviparous animals are required to allow these embryos to develop inside the maternal body, but must also eliminate an "invasion" by nonself. In colonial ascidians of the family Botryllidae, when two colonies are brought into contact at their growing edges, a hemolytic rejection reaction occurs between allogeneic colonies. Morula cells, a type of hemocyte, are the major effector cells in the hemolytic rejection. Morula cells infiltrate and aggregate where the two colonies make contact, and then discharge their vacuolar contents, which contain phenoloxidase and quinones. In viviparous botryllids, colonial contact at artificially cut surfaces always results in colonial fusion and establishment of a common vascular network even between allogeneic colonies in which the growing-edge contact results in rejection. This colonial fusion between incompatible colonies (surgical fusion) suggests that the allorecognition sites are not distributed in the vascular system in which the embryos are brooded. It is supposed that a common ancestor of the viviparous species lost the capacity for allorecognition in their vascular system to protect its embryos from alloreactivity, when it changed from ovoviviparous to viviparous in the course of evolution. The limited distribution of allorecognition sites would be a solution to the embryo-parent histoincompatibility in viviparity.     

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     

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.     

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.     

A complex allorecognition system in a reef-building coral: delayed responses,reversals and nontransitive hierarchies

A highly polymorphic and complex allorecognition system in the coral Stylophora pistillata was revealed in the field by assaying branch pair combinations among 11 colonies (181 assays) for 24 months. Replicates of between-colony combinations exhibited consistent outcomes, in both time scale and type of response. Different allogeneic combinations exhibited one of two main outcomes, either unilateral rejection, or an array of other incompatible reactions following a state of non-fusion. These responses were partially linked with color morphs (purple dominated yellow). An additional 22 isogeneic grafts resulted in complete fusion. Unilateral rejection occurred 1–7 months following initial contact. Nonfusion usually developed into skeletal suture barriers after 3–9 months, and then into unilateral colony-specific overgrowths at 6–23 months with some reversals in direction at 18–22 months. During this process, small lesions usually developed on the tissue of the subordinate partner, which were either overgrown by the dominant partner or healed. After two years, a network of overgrowths among colonies was established with essentially hierarchial properties, but some nontransitive interactions also occurred. The colonies segregated into three distinct histocompatibility groups; within each group, colonies engaged in nonfusion. Between groups, colonies exhibited nonfusion or rejected each other in a group-specific manner. Based on the results, we discuss the terminology used for fusion versus rejection phenomena in scleractinian corals, the possible genetic background for self-nonself recognition in Stylophora, and the methodological artifacts associated with the use of short-term allorecognition assays. Correspondence to: B. Rinkevich     

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.     

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.     

The social evolution of somatic fusion

The widespread potential for somatic fusion among different conspecific multicellular individuals suggests that such fusion is adaptive. However, because recognition of non-kin (allorecognition) usually leads to a rejection response, successful somatic fusion is limited to close kin. This is consistent with kin-selection theory, which predicts that the potential cost of fusion and the potential for somatic parasitism decrease with increasing relatedness. Paradoxically, however, Crozier found that, in the short term, positive-frequency-dependent selection eliminates the required genetic polymorphism at allorecognition loci. The 'Crozier paradox' may be solved if allorecognition is based on extrinsically balanced polymorphisms, for example at immune loci. Alternatively, the assumption of most models that self fusion is mutually beneficial is wrong. If fusion is on average harmful, selection will promote unconditional rejection. However, we propose that fusion within individuals is beneficial, selecting for the ability to fuse, but fusion between individuals on average costly, selecting for non-self recognition (rather than non-kin recognition). We discuss experimental data on fungi that are consistent with this hypothesis.     

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.     

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.     

Kin or self‐recognition? Colonial fusibility of the bryozoan Celleporella hyalina

We estimated fusion frequency with respect to coancestry in the bryozoan Celleporella hyalina, whose briefly planktonic sexually produced larvae settle on algal substrata and proceed to form encrusting colonies by iterative budding. Frequency of fusion between paired colonies growing on an artificial substratum was positively correlated with coefficient of relatedness, with allorecognition ability increasing during the early stages of colonial growth after larval settlement. Parents repressed the growth of F1 progeny with which they had fused. The results are concordant with the Feldgarden-Yund model of selection for self-recognition, which regards fusion with kin as an inevitable source of error whose cost diminishes with increasing relatedness. Contrary to fusion compatibility, gametic compatibility is negatively correlated with coancestry, indicating a need for further research on the possibility of common or linked genetic control that has opposite effect at somatic and gametic levels.     

EVIDENCE FOR SELECTION ON A CHORDATE HISTOCOMPATIBILITY LOCUS

Allorecognition is the ability of an organism to differentiate self or close relatives from unrelated individuals. The best known applications of allorecognition are the prevention of inbreeding in hermaphroditic species (e.g., the self‐incompatibility [SI] systems in plants), the vertebrate immune response to foreign antigens mediated by MHC loci, and somatic fusion, where two genetically independent individuals physically join to become a chimera. In the few model systems where the loci governing allorecognition outcomes have been identified, the corresponding proteins have exhibited exceptional polymorphism. But information about the evolution of this polymorphism outside MHC is limited. We address this subject in the ascidian Botryllus schlosseri, where allorecognition outcomes are determined by a single locus, called FuHC (Fusion/HistoCompatibility). Molecular variation in FuHC is distributed almost entirely within populations, with very little evidence for differentiation among different populations. Mutation plays a larger role than recombination in the creation of FuHC polymorphism. A selection statistic, neutrality tests, and distribution of variation within and among different populations all provide evidence for selection acting on FuHC, but are not in agreement as to whether the selection is balancing or directional.     

Isolation and Characterization of Schizosaccharomyces Pombe Mutants Affected in Mitotic Recombination

A haploid Schizosaccharomyces pombe strain carrying a heteroallelic duplication of the ade6 gene was used to isolate mitotic recombination-deficient mutants. Recombination between the different copies of the ade6 gene can lead to Ade+ segregants. These are observed as growing papillae when colonies of a suitable size are replicated onto selective medium. We isolated mutants which show an altered papillation phenotype. With two exceptions, they exhibit a decrease in the frequency of mitotic recombination between the heteroalleles of the duplication. The two other mutants display a hyper-recombination phenotype. The 12 mutations were allocated to at least nine distinct loci by recombination tests. Of the eight rec mutants analyzed further, six were also affected in mitotic intergenic recombination in the intervals cen2-mat or cen3-arg 1. No effect on mitotic intragenic recombination was observed. These data suggest that mitotic gene conversion and crossing over can be separated mutationally. Meiotic recombination occurs at the wild-type frequency in all mutants investigated.     

Molecular and physiological analysis of Arabidopsis mutants exhibiting altered sensitivities to aluminium

t Arabidopsis mutants with altered Al sensitivities have been isolated with the goal of identifying genes important for Al resistance and toxicity. By screening in a high-Al environment, seven Al-resistant mutants (alr) were isolated. The alr mutants were semi-dominant and constitute at least two unique loci in Arabidopsis. Nine Al-sensitive mutants (als) were also isolated. Complementation analysis revealed that of the nine als mutants, eight represent unique loci, indicating that Al sensitivity is genetically complex in Arabidopsis. The characterization of mutants with altered Al sensitivity will provide greater insights into the mechanisms of Al toxicity and resistance on a molecular and biochemical level.