Localization of Multiple Jellyfish Toxins Shows Specificity for Functionally Distinct Polyps and Nematocyst Types in a Colonial Hydrozoan.

Hydractinia symbiolongicarpus is a colonial hydrozoan that displays a division of labor through morphologically distinct and functionally specialized polyp types. As with all cnidarians, their venoms are housed in nematocysts, which are scattered across an individual. Here, we investigate the spatial distribution of a specific protein family, jellyfish toxins, in which multiple paralogs are differentially expressed across the functionally specialized polyps. Jellyfish toxins (JFTs) are known pore-forming toxins in the venoms of medically relevant species such as box jellyfish (class Cubozoa), but their role in other medusozoan venoms is less clear. Utilizing a publicly available single-cell dataset, we confirmed that four distinct H. symbiolongicarpus JFT paralogs are expressed in nematocyst-associated clusters, supporting these as true venom components in H. symbiolongicarpus. In situ hybridization and immunohistochemistry were used to localize the expression of these JFTs across the colony. These expression patterns, in conjunction with known nematocyst type distributions, suggest that two of these JFTs, HsymJFT1c-I and HsymJFT1c-II, are localized to specific types of nematocysts. We further interpret JFT expression patterns in the context of known regions of nematogenesis and differential rates of nematocyst turnover. Overall, we show that JFT expression patterns in H. symbiolongicarpus are consistent with the subfunctionalization of JFT paralogs across a partitioned venom system within the colony, such that each JFT is expressed within a specific set of functionally distinct polyp types and, in some cases, specific nematocyst types.

siRNA-mediated gene knockdown via electroporation in hydrozoan jellyfish embryos.

As the sister group to bilaterians, cnidarians stand in a unique phylogenetic position that provides insight into evolutionary aspects of animal development, physiology, and behavior. While cnidarians are classified into two types, sessile polyps and free-swimming medusae, most studies at the cellular and molecular levels have been conducted on representative polyp-type cnidarians and have focused on establishing techniques of genetic manipulation. Recently, gene knockdown by delivery of short hairpin RNAs into eggs via electroporation has been introduced in two polyp-type cnidarians, Nematostella vectensis and Hydractinia symbiolongicarpus, enabling systematic loss-of-function experiments. By contrast, current methods of genetic manipulation for most medusa-type cnidarians, or jellyfish, are quite limited, except for Clytia hemisphaerica, and reliable techniques are required to interrogate function of specific genes in different jellyfish species. Here, we present a method to knock down target genes by delivering small interfering RNA (siRNA) into fertilized eggs via electroporation, using the hydrozoan jellyfish, Clytia hemisphaerica and Cladonema paciificum. We show that siRNAs targeting endogenous GFP1 and Wnt3 in Clytia efficiently knock down gene expression and result in known planula phenotypes: loss of green fluorescence and defects in axial patterning, respectively. We also successfully knock down endogenous Wnt3 in Cladonema by siRNA electroporation, which circumvents the technical difficulty of microinjecting small eggs. Wnt3 knockdown in Cladonema causes gene expression changes in axial markers, suggesting a conserved Wnt/ß-catenin-mediated pathway that controls axial polarity during embryogenesis. Our gene-targeting siRNA electroporation method is applicable to other animals, including and beyond jellyfish species, and will facilitate the investigation and understanding of myriad aspects of animal development.

Specific Activities of Hydromedusan Ca2+ -Regulated Photoproteins.

Nowadays the recombinant Ca2+ -regulated photoproteins originating from marine luminous organisms are widely applied to monitor calcium transients in living cells due to their ability to emit light on Ca2+ binding. Here we report the specific activities of the recombinant Ca2+ -regulated photoproteins-aequorin from Aequorea victoria, obelins from Obelia longissima and Obelia geniculata, clytin from Clytia gregaria and mitrocomin from Mitrocoma cellularia. We demonstrate that along with bioluminescence spectra, kinetics of light signals and sensitivities to calcium, these photoproteins also differ in specific activities and consequently in quantum yields of bioluminescent reactions. The highest specific activities were found for obelins and mitrocomin, whereas those of aequorin and clytin were shown to be lower. To determine the factors influencing the variations in specific activities the fluorescence quantum yields for Ca2+ -discharged photoproteins were measured and found to be quite different varying in the range of 0.16-0.36. We propose that distinctions in specific activities may result from different efficiencies of singlet excited state generation and different fluorescence quantum yields of coelenteramide bound within substrate-binding cavity. This in turn may be conditioned by variations in the amino acid environment of the substrate-binding cavities and hydrogen bond distances between key residues and atoms of 2-hydroperoxycoelenterazine.

Cellular Reprogramming and Immortality: Expression Profiling Reveals Putative Genes Involved in Turritopsis dohrnii's Life Cycle Reversal.

To gather insight on the genetic network of cell reprogramming and reverse development in a nonmodel cnidarian system, we produced and annotated a transcriptome of the hydrozoan Turritopsis dohrnii, whose medusae respond to damage or senescence by metamorphosing into a juvenile stage (the polyp), briefly passing through an intermediate and uncharacterized stage (the cyst), where cellular transdifferentiation occurs. We conducted sequential and pairwise differential gene expression (DGE) analyses of the major life cycle stages involved in the ontogenetic reversal of T. dohrnii. Our DGE analyses of sequential stages of T. dohrnii's life cycle stages show that novel and characterized genes associated with aging/lifespan, regulation of transposable elements, DNA repair, and damage response, and Ubiquitin-related processes, among others, were enriched in the cyst stage. Our pairwise DGE analyses show that, when compared with the colonial polyp, the medusa is enriched with genes involved in membrane transport, the nervous system, components of the mesoglea, and muscle contraction, whereas genes involved in chitin metabolism and the formation of the primary germ layers are suppressed. The colonial polyp and reversed polyp (from cyst) show significant differences in gene expression. The reversed polyp is enriched with genes involved in processes such as chromatin remodeling and organization, matrix metalloproteinases, and embryonic development whereas suppressing genes involved in RAC G-protein signaling pathways. In summary, we identify genetic networks potentially involved in the reverse development of T. dohrnii and produce a transcriptome profile of all its life cycle stages, and paving the way for its use as a system for research on cell reprogramming.

Aequorea's secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing.

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

Pattern regulation in a regenerating jellyfish.

Jellyfish, with their tetraradial symmetry, offer a novel paradigm for addressing patterning mechanisms during regeneration. Here we show that an interplay between mechanical forces, cell migration and proliferation allows jellyfish fragments to regain shape and functionality rapidly, notably by efficient restoration of the central feeding organ (manubrium). Fragmentation first triggers actomyosin-powered remodeling that restores body umbrella shape, causing radial smooth muscle fibers to converge around 'hubs' which serve as positional landmarks. Stabilization of these hubs, and associated expression of Wnt6, depends on the configuration of the adjoining muscle fiber 'spokes'. Stabilized hubs presage the site of the manubrium blastema, whose growth is Wnt/ß-catenin dependent and fueled by both cell proliferation and long-range cell recruitment. Manubrium morphogenesis is modulated by its connections with the gastrovascular canal system. We conclude that body patterning in regenerating jellyfish emerges mainly from local interactions, triggered and directed by the remodeling process.

сWnt signaling modulation results in a change of the colony architecture in a hydrozoan.

At the polyp stage, most hydrozoan cnidarians form highly elaborate colonies with a variety of branching patterns, which makes them excellent models for studying the evolutionary mechanisms of body plan diversification. At the same time, molecular mechanisms underlying the robust patterning of the architecturally complex hydrozoan colonies remain unexplored. Using non-model hydrozoan Dynamena pumila we showed that the key components of the Wnt/ß-catenin (cWnt) pathway (ß-catenin, TCF) and the cWnt-responsive gene, brachyury 2, are involved in specification and patterning of the developing colony shoots. Strikingly, pharmacological modulation of the cWnt pathway leads to radical modification of the monopodially branching colony of Dynamena which acquire branching patterns typical for colonies of other hydrozoan species. Our results suggest that modulation of the cWnt signaling is the driving force promoting the evolution of the vast variety of the body plans in hydrozoan colonies and offer an intriguing possibility that the involvement of the cWnt pathway in the regulation of branching morphogenesis might represent an ancestral feature predating the cnidarian-bilaterian split.

Molecular characterisation of a cellular conveyor belt in Clytia medusae.

The tentacular system of Clytia hemisphaerica medusa (Cnidaria, Hydrozoa) has recently emerged as a promising experimental model to tackle the developmental mechanisms that regulate cell lineage progression in an early-diverging animal phylum. From a population of proximal stem cells, the successive steps of tentacle stinging cell (nematocyte) elaboration, are spatially ordered along a "cellular conveyor belt". Furthermore, the C. hemisphaerica tentacular system exhibits bilateral organisation, with two perpendicular polarity axes (proximo-distal and oral-aboral). We aimed to improve our knowledge of this cellular system by combining RNAseq-based differential gene expression analyses and expression studies of Wnt signalling genes. RNAseq comparisons of gene expression levels were performed (i) between the tentacular system and a control medusa deprived of all tentacles, nematogenic sites and gonads, and (ii) between three samples staggered along the cellular conveyor belt. The behaviour in these differential expression analyses of two reference gene sets (stem cell genes; nematocyte genes), as well as the relative representations of selected gene ontology categories, support the validity of the cellular conveyor belt model. Expression patterns obtained by in situ hybridisation for selected highly differentially expressed genes and for Wnt signalling genes are largely consistent with the results from RNAseq. Wnt signalling genes exhibit complex spatial deployment along both polarity axes of the tentacular system, with the Wnt/ß-catenin pathway probably acting along the oral-aboral axis rather than the proximo-distal axis. These findings reinforce the idea that, despite overall radial symmetry, cnidarians have a full potential for elaboration of bilateral structures based on finely orchestrated deployment of an ancient developmental gene toolkit.

Metabolic Activation and Scaling in Two Species of Colonial Cnidarians.

Metabolic activation can have a profound impact, for instance, by more than compensating for the lower resting metabolic rates of large organisms compared to smaller ones. In some animals, activity can easily be judged by the rate of muscle-driven movement. In sessile organisms, however, judging activity is less straightforward, although feeding often results in metabolic activation. Two colonial cnidarians were examined in this context, using entirely lab-grown material to remove any artifactual effects of experimental manipulations. Hydractinia symbiolongicarpus is a carnivorous hydroid that uses active muscular contractions to drive its gastrovascular fluid. Sympodium sp., on the other hand, is an octocoral that hosts photosynthetic Symbiodinium and uses cilia to propel its gastrovascular fluid. Measures of oxygen uptake indicated that feeding activated metabolism in H. symbiolongicarpus. While light treatment had no effect on subsequent dark metabolism in Sympodium sp., stress activated metabolism to an extent comparable to H. symbiolongicarpus. In both taxa, different individual size measures or synthetic size measures derived from principal component analysis produced different scaling relationships between metabolism and size. On balance, the data suggest that scaling was negatively allometric in Sympodium sp. and nearly isometric in H. symbiolongicarpus; yet metabolic activation was comparable in the two species. Regardless of the size measure used, active and resting colonies of H. symbiolongicarpus exhibited similar scaling relationships. Colonial animals may lack the large difference between resting and active metabolic rates found in highly active animals, and this may be related to how their metabolism scales with size.

Comparative Analysis of the Soluble Proteome and the Cytolytic Activity of Unbleached and Bleached Millepora complanata ("Fire Coral") from the Mexican Caribbean.

Coral bleaching caused by global warming has resulted in massive damage to coral reefs worldwide. Studies addressing the consequences of elevated temperature have focused on organisms of the class Anthozoa, and up to now, there is little information regarding the mechanisms by which reef forming Hydrozoans face thermal stress. In this study, we carried out a comparative analysis of the soluble proteome and the cytolytic activity of unbleached and bleached Millepora complanata ("fire coral") that inhabited reef colonies exposed to the 2015-2016 El Niño-Southern Oscillation in the Mexican Caribbean. A differential proteomic response involving proteins implicated in key cellular processes, such as glycolysis, DNA repair, stress response, calcium homeostasis, exocytosis, and cytoskeleton organization was found in bleached hydrocorals. Four of the proteins, whose levels increased in bleached specimens, displayed sequence similarity to a phospholipase A2, an astacin-like metalloprotease, and two pore forming toxins. However, a protein, which displayed sequence similarity to a calcium-independent phospholipase A2, showed lower levels in bleached cnidarians. Accordingly, the hemolytic effect of the soluble proteome of bleached hydrocorals was significantly higher, whereas the phospholipase A2 activity was significantly reduced. Our results suggest that bleached M. complanata is capable of increasing its toxins production in order to balance the lack of nutrients supplied by its symbionts.

Tuning the fluorescence of calcium-discharged photoprotein obelin via mutating at the His22-Phe88-Trp92 triad - a QM/MM study.

The fluorescence (FL) of calcium-discharged photoprotein (CaDP) can be altered by easily mutating CaDP without modifying coelenteramide (CLM), which is the decarboxylation product of coelenterazine in calcium-regulated photoprotein. The His22-Phe88-Trp92 triad (the ordering numbers of three amino acids are sorted by a crystal structure (PDB: 2F8P) of calcium-discharged obelin, i.e., CaDP-obelin) is closely related to CaDP-obelin FL, since it exists in close proximity to the 5-p-hydroxyphenyl of CLM. Therefore, it is important to thoroughly investigate how the mutations of this triad affect the emission color of CaDP-obelin FL. In this study, by mutating wild-type CaDP-obelin (WT) at the His22-Phe88-Trp92 triad, we theoretically constructed its nine mutants of separable FL colors. Through combined quantum mechanics and molecular mechanics (QM/MM) calculations and molecular dynamics (MD) simulations, the influence of the mutations of this triad on the CaDP-obelin FL was analyzed considering the H-bond effect and the charge effect. This study demonstrated that the mutations at the His22-Phe88-Trp92 triad redistribute the charges on the D-π-A molecule, CLM, change the charge transfer from the D to the (π + A) moiety, and thereby alter the FL emission. Appending more negative charges on the phenolate moiety of CLM benefits the FL redshift.

High doses of CRISPR/Cas9 ribonucleoprotein efficiently induce gene knockout with low mosaicism in the hydrozoan Clytia hemisphaerica through microhomology-mediated deletion.

Targeted mutagenesis using CRISPR/Cas9 technology has been shown to be a powerful approach to examine gene function in diverse metazoan species. One common drawback is that mixed genotypes, and thus variable phenotypes, arise in the F0 generation because incorrect DNA repair produces different mutations amongst cells of the developing embryo. We report here an effective method for gene knockout (KO) in the hydrozoan Clytia hemisphaerica, by injection into the egg of Cas9/sgRNA ribonucleoprotein complex (RNP). Expected phenotypes were observed in the F0 generation when targeting endogenous GFP genes, which abolished fluorescence in embryos, or CheRfx123 (that codes for a conserved master transcriptional regulator for ciliogenesis) which caused sperm motility defects. When high concentrations of Cas9 RNP were used, the mutations in target genes at F0 polyp or jellyfish stages were not random but consisted predominantly of one or two specific deletions between pairs of short microhomologies flanking the cleavage site. Such microhomology-mediated (MM) deletion is most likely caused by microhomology-mediated end-joining (MMEJ), which may be favoured in early stage embryos. This finding makes it very easy to isolate uniform, largely non-mosaic mutants with predictable genotypes in the F0 generation in Clytia, allowing rapid and reliable phenotype assessment.

Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: An update for 2013-2014.

This review is the eighth update of the original article published in 1999 on the application of Matrix-assisted laser desorption/ionization mass spectrometry (MALDI) mass spectrometry to the analysis of carbohydrates and glycoconjugates and brings coverage of the literature to the end of 2014. Topics covered in the first part of the review include general aspects such as theory of the MALDI process, matrices, derivatization, MALDI imaging, fragmentation, and arrays. The second part of the review is devoted to applications to various structural types such as oligo- and poly- saccharides, glycoproteins, glycolipids, glycosides, and biopharmaceuticals. Much of this material is presented in tabular form. The third part of the review covers medical and industrial applications of the technique, studies of enzyme reactions, and applications to chemical synthesis. © 2018 Wiley Periodicals, Inc. Mass Spec Rev 37:353-491, 2018.

Acid-Tolerant Monomeric GFP from Olindias formosa.

The fluorescent protein (FP) color palette has greatly contributed to the visualization of molecular and cellular processes. However, most FPs lose fluorescence at a pH lower than their neutral pKa (∼6), and this has hampered their application in acidic organelles (pH ∼4.5-6.0). Currently, several cyan- and red-colored acid-tolerant FPs are available; however, there are few reports of acid-tolerant green FPs (GFPs) that are practically applicable to bioimaging. Here, we developed the acid-tolerant monomeric GFP "Gamillus" from the jellyfish Olindias formosa, with excellent brightness, maturation speed, and photostability. Results from X-ray crystallography and point mutagenesis suggest that across a broad pH range the acid tolerance is attributed to stabilization of deprotonation in the chromophore phenyl ring by forming a unique trans configuration. We demonstrate that Gamillus can serve as a molecular tag suitable for imaging in acidic organelles through autophagy-mediated molecular tracking to lysosomes.

A safer, urea-based in situ hybridization method improves detection of gene expression in diverse animal species.

In situ hybridization is a widely employed technique allowing spatial visualization of gene expression in fixed specimens. It has greatly advanced our understanding of biological processes, including developmental regulation. In situ protocols are today routinely followed in numerous laboratories, and although details might change, they all include a hybridization step, where specific antisense RNA or DNA probes anneal to the target nucleic acid sequence. This step is generally carried out at high temperatures and in a denaturing solution, called hybridization buffer, commonly containing 50% (v/v) formamide - a hazardous chemical. When applied to the soft-bodied hydrozoan medusa Clytia hemisphaerica, we found that this traditional hybridization approach was not fully satisfactory, causing extensive deterioration of morphology and tissue texture which compromised our observation and interpretation of results. We thus tested alternative solutions for in situ detection of gene expression and, inspired by optimized protocols for Northern and Southern blot analysis, we substituted the 50% formamide with an equal volume of 8M urea solution in the hybridization buffer. Our new protocol not only yielded better morphologies and tissue consistency, but also notably improved the resolution of the signal, allowing more precise localization of gene expression and reducing aspecific staining associated with problematic areas. Given the improved results and reduced manipulation risks, we tested the urea protocol on other metazoans, two brachiopod species (Novocrania anomala and Terebratalia transversa) and the priapulid worm Priapulus caudatus, obtaining a similar reduction of aspecific probe binding. Overall, substitution of formamide by urea during in situ hybridization offers a safer alternative, potentially of widespread use in research, medical and teaching contexts. We encourage other workers to test this approach on their study organisms, and hope that they will also obtain better sample preservation, more precise expression patterns and fewer problems due to aspecific staining, as we report here for Clytia medusae and Novocrania and Terebratalia developing larvae.

Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca2+ -regulated Photoproteins of Different Organisms.

Upon binding their metal ion cofactors, Ca2+ -regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca2+ -regulated photoproteins-aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata-demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca2+ -regulated photoproteins.

Intramolecular interactions that control voltage sensitivity in the jShak1 potassium channel from Polyorchis penicillatus.

Voltage-gated potassium ion (Kv) channel proteins respond to changes in membrane potential by changing the probability of K+ flux through an ion-selective pore. Kv channels from different paralogous and orthologous families have widely varying V50 values. The voltage-sensing transmembrane helices (S4) of different channels contain four to seven basic residues that are responsible for transducing changes in transmembrane potential into the energy required to shift the equilibrium between the open- and closed-channel conformations. These residues also form electrostatic interaction networks with acidic residues in the S2 and S3 helices that stabilize the open and the closed states to different extents. The length and composition of the extracellular loop connecting the S3 and S4 helices (S3-S4 loop) also shape the voltage response. We describe mutagenesis experiments on the jellyfish (Polyorchis penicillatus) Kv1 family jShak1 channel to evaluate how variants of the S3-S4 loop affect the voltage sensitivity of this channel. In combination with changes in the length and composition of the S3-S4 linker, we mutated a residue on the S2 helix (N227) that in most Kv1 family channels is glutamate (E226 in mouse Kv1.2, E283 in D. melanogaster Shaker). Some individual loop replacement mutants cause major changes in voltage sensitivity, depending on a combination of length and composition. Pairwise combinations of the loop mutations and the S2 mutations interact to yield quantitatively distinct, non-additive changes in voltage sensitivity. We conclude that the S3-S4 loop interacts energetically with the residue at position N227 during the transitions between open and closed states of the channel.

Crystal structure of the cyan fluorescent protein Cerulean-S175G.

Enhanced cyan fluorescent protein (ECFP) was derived from Aequorea victoria green fluorescent protein (avGFP), notably with S65T/Y66W mutations. Its chromophore consists of a tripeptide comprised of Thr65, Trp66 and Gly67 (TWG) residues, while that of avGFP consists of a Ser65, Tyr66 and Gly67 (SYG) tripeptide. Cerulean and SCFP3A were derived from ECFP-S72A/H148D (a double mutation) with additional Y145A and S175G mutations, respectively, while Cerulean-S175G has both mutations (Y145A and S175G). The crystal structures of these ECFP variants at neutral pH were reported to adopt two distinct major conformations called ECFP and Cerulean. In this study, Cerulean-S175G was revealed to adopt only the Cerulean conformation, while Cerulean has been reported to adopt both the ECFP and the Cerulean conformations in its crystal structures. Sharing the same S175G mutation with SCFP3A, Cerulean-S175G showed a slightly increased quantum yield, like SCFP3A, but did not adopt the ECFP conformation adopted by SCFP3A. Detailed comparison of Cerulean-S175G and other ECFP variants revealed that the notable conformational changes in ECFP variants can be understood mainly in terms of the interaction between the Trp66 residue of the chromophore and residues 145-148 of ß-strand 7.

Role of certain amino acid residues of the coelenterazine-binding cavity in bioluminescence of light-sensitive Ca(2+)-regulated photoprotein berovin.

Bright bioluminescence of ctenophores is caused by Ca(2+)-regulated photoproteins. Although these photoproteins are functionally identical to and share many properties of cnidarian photoproteins, like aequorin and obelin, and retain the same spatial architecture, they are extremely sensitive to light, i.e. lose the ability to bioluminesce on exposure to light over the entire absorption spectrum. In addition, the degree of identity of their amino acid sequences with those of cnidarian photoproteins is only 29.4%. This suggests that the residues involved in bioluminescence of ctenophore and cnidarian photoproteins significantly differ. Here we describe the bioluminescent properties of berovin mutants with substitution of the residues located in the photoprotein internal cavity. Since the spatial structure of berovin bound with a substrate is not determined yet, to identify these residues we have modeled it with an accommodated substrate using the structures of some cnidarian Ca(2+)-regulated photoproteins with bound coelenterazine or coelenteramide as templates in order to obtain an adequate sampling and to take into account all possible conformers and variants for ligand-protein docking. Based on the impact of substitutions on the bioluminescent properties and model structures we speculate that within the internal cavity of ctenophore photoproteins, coelenterazine is bound as a 2-peroxy anion adduct which is stabilized owing to Coulomb interaction with a positively charged guanidinium group of Arg41 paired with Tyr204. In this case, the bioluminescence reaction is triggered by only calcium-induced conformational changes leading to the disturbance of charge-charge interaction.

Allorecognition proteins in an invertebrate exhibit homophilic interactions.

Sessile colonial invertebrates-animals such as sponges, corals, bryozoans, and ascidians-can distinguish between their own tissues and those of conspecifics upon contact [1]. This ability, called allorecognition, mediates spatial competition and can prevent stem cell parasitism by ensuring that colonies only fuse with self or close kin. In every taxon studied to date, allorecognition is controlled by one or more highly polymorphic genes [2-8]. However, in no case is it understood how the proteins encoded by these genes discriminate self from non-self. In the cnidarian Hydractinia symbiolongicarpus, allorecognition is controlled by at least two highly polymorphic allorecognition genes, Alr1 and Alr2 [3, 5, 9-12]. Sequence variation at each gene predicts allorecognition in laboratory strains such that colonies reject if they do not share a common allele at either locus, fuse temporarily if they share an allele at only one locus, or fuse permanently if they share an allele at both genes [5, 9]. Here, we show that the gene products of Alr1 and Alr2 (Alr1 and Alr2) are self-ligands with extraordinary specificity. Using an in vitro cell aggregation assay, we found that Alr1 and Alr2 bind to themselves homophilically across opposing cell membranes. For both proteins, each isoform bound only to itself or to an isoform of nearly identical sequence. These results provide a mechanistic explanation for the exquisite specificity of Hydractinia allorecognition. Our results also indicate that hydroids have evolved a molecular strategy of self-recognition that is unique among characterized allorecognition systems within and outside invertebrates.