Sea anemone model has a single Toll-like receptor that can function in pathogen detection, NF-κB signal transduction, and development

In organisms from insects to vertebrates, Toll-like receptors (TLRs) are primary pathogen detectors that activate downstream pathways, specifically those that direct expression of innate immune effector genes. TLRs also have roles in development in many species. The sea anemone Nematostella vectensis is a useful cnidarian model to study the origins of TLR signaling because its genome encodes a single TLR and homologs of many downstream signaling components, including the NF-κB pathway. We have characterized the single N. vectensis TLR (Nv-TLR) and demonstrated that it can activate canonical NF-κB signaling in human cells. Furthermore, we show that the intracellular Toll/IL-1 receptor (TIR) domain of Nv-TLR can interact with the human TLR adapter proteins MAL and MYD88. We demonstrate that the coral pathogen Vibrio coralliilyticus causes a rapidly lethal disease in N. vectensis and that heat-inactivated V. coralliilyticus and bacterial flagellin can activate a reconstituted Nv-TLR–to–NF-κB pathway in human cells. By immunostaining of anemones, we show that Nv-TLR is expressed in a subset of cnidocytes and that many of these Nv-TLR–expressing cells also express Nv-NF-κB. Additionally, the nematosome, which is a Nematostella-specific multicellular structure, expresses Nv-TLR and many innate immune pathway homologs and can engulf V. coralliilyticus. Morpholino knockdown indicates that Nv-TLR also has an essential role during early embryonic development. Our characterization of this primitive TLR and identification of a bacterial pathogen for N. vectensis reveal ancient TLR functions and provide a model for studying the molecular basis of cnidarian disease and immunity.IOS-1354935 - National Science Foundation (NSF); GRFP - National Science Foundation (NSF); GRFP - National Science Foundation (NSF); 1262934 - National Science Foundation (NSF); 2014-BSP - Arnold and Mabel Beckman Foundatio

Current directions and future perspectives from the third Nematostella research conference

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Zoology 118 (2015): 135-140, doi:10.1016/j.zool.2014.06.005.The third Nematostella vectensis Research Conference took place in December 2013 in Eilat, Israel, as a satellite to the 8th International Conference on Coelenterate Biology. The starlet sea anemone, Nematostella vectensis, has emerged as a powerful cnidarian model, in large part due to the extensive genomic and transcriptomic resources and molecular approaches that are becoming available for Nematostella, which were the focus of several presentations. In addition, research was presented highlighting the broader utility of this species for studies of development, circadian rhythms, signal transduction, and gene–environment interactions.Research in the authors’ laboratories on Nematostella is supported by National Science Foundation grants MCB-1057354 to A.M.T. and MCB-0924749 to T.D.G. Travel support for the meeting was provided to T.D.G. by Illumina, Inc. (San Diego, CA, USA), to A.M.R. by the University of North Carolina at Charlotte, and to A.M.T. by the Israel–US Binational Science Foundation (Jerusalem, Israel)

Microevolution of the oxidative stress response: organismal and transcriptomic effects of peroxide exposure in the starlet sea anemone Nematostella vectensis

The starlet sea anemone, Nematostella vectensis (phylum Cnidaria, class Anthozoa), inhabits tidal marshes along the Atlantic coast of North America. These shallow coastal habitats are subject to extreme environmental variability, including fluctuating levels of reactive oxygen species (ROS). High ROS concentrations can be cytotoxic and lead to oxidative stress. Given the importance of tidal marshes for marine biodiversity and their susceptibility to oxidative stress, it is important to study how resident organisms counteract oxidative stress. N. vectensis is an excellent model system for addressing the evolution of oxidative stress mechanisms because (1) this anemone exhibits tolerance to a variety of environmental stressors, (2) there is clear evidence of local adaptation to stress in different populations and sub-populations, and (3) protein-coding polymorphisms have been identified, some in proteins that are implicated in stress response. I exposed fifteen different clone lines of N. vectensis collected from four estuaries to biologically relevant levels of hydrogen peroxide. Pronounced differences are apparent between clone lines collected from Meadowlands, NJ, Baruch, SC, and Kingsport, NS, as well as among twelve clones collected at a single Cape Cod marsh. This is the first study that demonstrates intraspecific variation in the oxidative stress response of N. vectensis. To understand how the peroxide response of these clones might differ at the level of gene expression, I sequenced the transcriptomes of 7 clone lines under control conditions and when exposed to survivable levels of peroxide for 24 h. I found survivable peroxide exposure induces robust and repeatable changes in gene expression, including the up-regulation of genes shown to be associated with cellular responses to oxidative stress, thermal stress, and UV stress, and down-regulation of genes that promote generation of ROS, including the enzymes involved in oxidative phosphorylation. Additionally, I identified pronounced differences in the peroxide-induced transcriptional profiles between genets of N. vectensis. The existence of a conserved oxidative stress response has major implications for studies on how other cnidarians, particularly corals, will withstand the impacts of climate change. Indeed, the proximal cause of coral bleaching--the major threat to corals worldwide--is oxidative stress

SeaBase : a multispecies transcriptomic resource and platform for gene network inference

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Integrative and Comparative Biology 54 (2014): 250-263, doi: 10.1093/icb/icu065.Marine and aquatic animals are extraordinarily useful as models for identifying mechanisms of development and evolution, regeneration, resistance to cancer, longevity and symbiosis, among many other areas of research. This is due to the great diversity of these organisms and their wide-ranging capabilities. Genomics tools are essential for taking advantage of these “free lessons” of nature. However, genomics and transcriptomics are challenging in emerging model systems. Here, we present SeaBase, a tool for helping to meet these needs. Specifically, SeaBase provides a platform for sharing and searching transcriptome data. More importantly, SeaBase will support a growing number of tools for inferring gene network mechanisms. The first dataset available on SeaBase is a developmental transcriptome profile of the sea anemone Nematostella vectensis (Anthozoa, Cnidaria). Additional datasets are currently being prepared and we are aiming to expand SeaBase to include user-supplied data for any number of marine and aquatic organisms, thereby supporting many potentially new models for gene network studies.2015-06-0

Investigating alternative life history trajectories in two species of Edwardsiid sea anemones using ecological, transcriptomic, and molecular approaches

Life histories unfold within the ecological context of an organism's environment, and thus are intimately linked to organismal fitness. The evolution of alternate life history strategies, either within or between taxa, can profoundly affect ontogeny, ecology, and population dynamics. Many cnidarians (sea anemones, corals, jellyfish, etc.) exhibit complex life histories involving sexual reproduction and multiple modes of asexual reproduction. Sea anemones of the family Edwardsiidae exemplify this complexity, and are therefore an attractive system for studying the developmental and ecological ramifications of life history evolution. I used intra- and interspecific comparisons of two Edwardsiid anemones, Edwardsiella lineata, and Nematostella vectensis to investigate alternative life histories using a multifaceted approach that included field-based ecological surveys, functional genetics, transcriptomics, and phylogenetics. Both anemones are capable of sexual and asexual reproduction. N. vectensis produces a rapidly maturing direct developing larva. By contrast, E. lineata has evolved a new larval stage that parasitizes the ctenophore, Mnemiopsis leidyi. Through fieldwork surveys and laboratory culture, I documented several life history traits, such as a previously un-characterized, pre-parasitic larval stage, and the developmental dynamics of early-stage parasitic infections, that augmented gaps in our knowledge of E. lineata's life history. To better understand how and when E. lineata evolved its novel, parasitic life history, I worked with collaborators in the Finnerty lab to sequence, assemble and annotate the transcriptome. Through a multigene molecular clock approach, enabled by the E. lineata transcriptome assembly, I estimated the divergence date for these two anemones between 215-364 million years ago, thereby establishing an upper bound for the innovation of E. lineata's derived, parasitic life history. Testing a hypothesis that Wnt signaling, which patterns the oral-aboral (OA) axis during embryogenesis, also patterns the OA axis during regeneration, I demonstrated that canonical Wnt signaling is sufficient for oral tissue fate across alternate life histories (embryogenesis and regeneration) of N. vectensis. Taken together, these dissertation research activities constitute an integrative approach to investigating the evolution of life histories, and are a step towards establishing E. lineata and N. vectensis as models for studying the evolutionary developmental mechanisms of parasitism and regeneration

Insights into the early evolution of NF-kappaB signaling based on computational analyses of cnidarian genomes and transcriptomes

NF-kappaB is an ancient transcription factor that is known to play a central role in regulating cellular stress responses in vertebrates and insects, including the innate immune response, and the response to a range of physiochemical insults such as UV radiation and oxidative stress. The early evolution of this pathway is not well understood, because little is known about NF-kappaB signaling in so-called basal animal lineages (e.g., sponges, cnidarians) or closely related outgroups to the Metazoa. Key to understanding the function of a transcription factor is to identify the target genes whose transcription it regulates. To investigate the regulatory role of NF-kappaB in basal animals, specifically the sea anemone Nematostella vectensis, I developed ForSite, a computational tool that identifies putative transcription factor binding sites in the genome in proximity to expressed genes, and I helped to generate a new annotated reference transcriptome for N. vectensis. After demonstrating that ForSite could be used to identify a set of genes enriched for known NF-kappaB targets in human, I applied ForSite along with multiple winnowing criteria (co-localization of p300 binding; evolutionary conservation of target genes) to identify a high-priority list of potential NF-kappaB targets in the anemone. Among the most convincing set of likely target genes are members of a conserved anti-viral pathway, which suggests NF-kappaB plays an ancient role in innate immunity that dates to the cnidarian-bilaterian ancestor. Application of ForSite to two additional cnidarian species, Hydra magnipapillata and Acropora digitifera, failed to show significant conservation of regulation of biological processes by NF-kappaB among the cnidarian species

The evolution of the four subunits of voltage-gated calcium channels : ancient roots, increasing complexity, and multiple losses

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Genome Biology and Evolution 6 (2014): 2210-2217, doi:10.1093/gbe/evu177.The alpha subunits of voltage-gated calcium channels (Cavs) are large transmembrane proteins responsible for crucial physiological processes in excitable cells. They are assisted by three auxiliary subunits that can modulate their electrical behavior. Little is known about the evolution and roles of the various subunits of Cavs in nonbilaterian animals and in nonanimal lineages. For this reason, we mapped the phyletic distribution of the four channel subunits and reconstructed their phylogeny. Although alpha subunits have deep evolutionary roots as ancient as the split between plants and opistokonths, beta subunits appeared in the last common ancestor of animals and their close-relatives choanoflagellates, gamma subunits are a bilaterian novelty and alpha2/delta subunits appeared in the lineage of Placozoa, Cnidaria, and Bilateria. We note that gene losses were extremely common in the evolution of Cavs, with noticeable losses in multiple clades of subfamilies and also of whole Cav families. As in vertebrates, but not protostomes, Cav channel genes duplicated in Cnidaria. We characterized by in situ hybridization the tissue distribution of alpha subunits in the sea anemone Nematostella vectensis, a nonbilaterian animal possessing all three Cav subfamilies common to Bilateria. We find that some of the alpha subunit subtypes exhibit distinct spatiotemporal expression patterns. Further, all six sea anemone alpha subunit subtypes are conserved in stony corals, which separated from anemones 500 MA. This unexpected conservation together with the expression patterns strongly supports the notion that these subtypes carry unique functional roles

The evolution of the four subunits of voltage-gated calcium channels : ancient roots, increasing complexity, and multiple losses

© The Author(s), 2014. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Genome Biology and Evolution 6 (2014): 2210-2217, doi:10.1093/gbe/evu177.The alpha subunits of voltage-gated calcium channels (Cavs) are large transmembrane proteins responsible for crucial physiological processes in excitable cells. They are assisted by three auxiliary subunits that can modulate their electrical behavior. Little is known about the evolution and roles of the various subunits of Cavs in nonbilaterian animals and in nonanimal lineages. For this reason, we mapped the phyletic distribution of the four channel subunits and reconstructed their phylogeny. Although alpha subunits have deep evolutionary roots as ancient as the split between plants and opistokonths, beta subunits appeared in the last common ancestor of animals and their close-relatives choanoflagellates, gamma subunits are a bilaterian novelty and alpha2/delta subunits appeared in the lineage of Placozoa, Cnidaria, and Bilateria. We note that gene losses were extremely common in the evolution of Cavs, with noticeable losses in multiple clades of subfamilies and also of whole Cav families. As in vertebrates, but not protostomes, Cav channel genes duplicated in Cnidaria. We characterized by in situ hybridization the tissue distribution of alpha subunits in the sea anemone Nematostella vectensis, a nonbilaterian animal possessing all three Cav subfamilies common to Bilateria. We find that some of the alpha subunit subtypes exhibit distinct spatiotemporal expression patterns. Further, all six sea anemone alpha subunit subtypes are conserved in stony corals, which separated from anemones 500 MA. This unexpected conservation together with the expression patterns strongly supports the notion that these subtypes carry unique functional roles

Ultraviolet radiation significantly enhances the molecular response to dispersant and sweet crude oil exposure in Nematostella vectensis

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Marine Environmental Research 134 (2018): 96-108, doi:10.1016/j.marenvres.2018.01.002.Estuarine organisms are subjected to combinations of anthropogenic and natural stressors, which together can reduce an organisms' ability to respond to either stress or can potentiate or synergize the cellular impacts for individual stressors. Nematostella vectensis (starlet sea anemone) is a useful model for investigating novel and evolutionarily conserved cellular and molecular responses to environmental stress. Using RNA-seq, we assessed global changes in gene expression in Nematostella in response to dispersant and/or sweet crude oil exposure alone or combined with ultraviolet radiation (UV). A total of 110 transcripts were differentially expressed by dispersant and/or crude oil exposure, primarily dominated by the down-regulation of 74 unique transcripts in the dispersant treatment. In contrast, UV exposure alone or combined with dispersant and/or oil resulted in the differential expression of 1133 transcripts, of which 436 were shared between all four treatment combinations. Most significant was the differential expression of 531 transcripts unique to one or more of the combined UV/chemical exposures. Main categories of genes affected by one or more of the treatments included enzymes involved in xenobiotic metabolism and transport, DNA repair enzymes, and general stress response genes conserved among vertebrates and invertebrates. However, the most interesting observation was the induction of several transcripts indicating de novo synthesis of mycosporine-like amino acids and other novel cellular antioxidants. Together, our data suggest that the toxicity of oil and/or dispersant and the complexity of the molecular response are significantly enhanced by UV exposure, which may co-occur for shallow water species like Nematostella.This material is based upon work supported by the National Science Foundation under Grant No. MCB1057152 (MJJ), MCB1057354 (AMT) and DEB1545539 (AMR)

The Birth and Death of Toxins with Distinct Functions: A Case Study in the Sea Anemone Nematostella

The cnidarian Nematostella vectensis has become an established lab model, providing unique opportunities for venom evolution research. The Nematostella venom system is multimodal: involving both nematocytes and ectodermal gland cells, which produce a toxin mixture whose composition changes throughout the life cycle. Additionally, their modes of interaction with predators and prey vary between eggs, larvae, and adults, which is likely shaped by the dynamics of the venom system. Nv1 is a major component of adult venom, with activity against arthropods (through specific inhibition of sodium channel inactivation) and fish. Nv1 is encoded by a cluster of at least 12 nearly identical genes that were proposed to be undergoing concerted evolution. Surprisingly, we found that Nematostella venom includes several Nv1 paralogs escaping a pattern of general concerted evolution, despite belonging to the Nv1-like family. Here, we show two of these new toxins, Nv4 and Nv5, are lethal for zebrafish larvae but harmless to arthropods, unlike Nv1. Furthermore, unlike Nv1, the newly identified toxins are expressed in early life stages. Using transgenesis and immunostaining, we demonstrate that Nv4 and Nv5 are localized to ectodermal gland cells in larvae. The evolution of Nv4 and Nv5 can be described either as neofunctionalization or as subfunctionalization. Additionally, the Nv1-like family includes several pseudogenes being an example of nonfunctionalization and venom evolution through birth-and-death mechanism. Our findings reveal the evolutionary history for a toxin radiation and point toward the ecological function of the novel toxins constituting a complex cnidarian venom.publishedVersio