James C. Sullivan

StellaBase: The Nematostella vectensis Genomics Database

StellaBase, the Nematostella vectensis Genomics Database, is a web-based resource that will facilitate desktop and bench-top studies of the starlet sea anemone. Nematostella is an emerging model organism that has already proven useful for addressing fundamental questions in developmental evolution and evolutionary genomics. StellaBase allows users to query the assembled Nematostella genome, a confirmed gene library, and a predicted genome using both keyword and homology based search functions. Data provided by these searches will elucidate gene family evolution in early animals. Unique research tools, including a Nematostella genetic stock library, a primer library, a literature repository and a gene expression library will provide support to the burgeoning Nematostella research community. The development of StellaBase accompanies significant upgrades to CnidBase, the Cnidarian Evolutionary Genomics Database. With the completion of the first sequenced cnidarian genome, genome comparison tools have been added to CnidBase. In addition, StellaBase provides a framework for the integration of additional species-specific databases into CnidBase. StellaBase is available at http://www.stellabase.org.

Rel homology domain-containing transcription factors in the cnidarian Nematostella vectensis.

The Rel/NF-κB and NFAT families of transcription factors are related through an N-terminal DNA-binding domain called the Rel Homology domain (RHD). Neither the RHD nor the NF-κB pathway has been identified in a basal (i.e., nonbilaterian) animal phylum. Using genomic and cDNA databases, we have identified two RHD domain-containing proteins from the cnidarian Nematostella vectensis: an NF-κB-like protein (Nv-NF-κB) and an NFAT-like protein (Nv-NFAT). The gene structure and RHD predicted amino acid sequence of Nv-nfkb are similar to those of the vertebrate NF-κB p50/p52 proteins, whereas the sequence of Nv-NFAT allows only ambiguous assignment to the NFAT family. Nv-NF-κB lacks the C-terminal IκB-like sequences present in all other NF-κB proteins. There are, however, two IκB-like genes in Nematostella encoded by loci distinct from Nv-nfkb. The separate nfkb and ikb genes of Nematostella may reflect the ancestral metazoan condition, suggesting that a gene fusion event created the nfkb genes in Drosophila and vertebrates. Nematostella also has genes that encode upstream and downstream components of the vertebrate NF-κB signaling pathway. Upstream components include Toll- and tumor necrosis-like receptors and ligands, adaptor proteins (Trafs, Myd88), caspases, and a TBK-like kinase. Downstream components include the NF-κB coactivator protein Bcl-3 and several NF-κB target genes. These results demonstrate that RHD-containing transcription factors and associated pathways are evolutionarily more ancient than previously known. Moreover, they suggest models for the evolutionary diversification of the insect and vertebrate Rel/NF-κB/IκB and NFAT gene families and suggest that cnidarians possess an NF-κB-regulated developmental or stress response pathway.

Genomic Survey of Candidate Stress-Response Genes in the Estuarine Anemone Nematostella vectensis

Salt marshes are challenging habitats due to natural variability in key environmental parameters including temperature, salinity, ultraviolet light, oxygen, sulfides, and reactive oxygen species. Compounding this natural variation, salt marshes are often heavily impacted by anthropogenic insults including eutrophication, toxic contamination, and coastal development that alter tidal and freshwater inputs. Commensurate with this environmental variability, estuarine animals generally exhibit broader physiological tolerances than freshwater, marine, or terrestrial species. One factor that determines an organisms physiological tolerance is its ability to upregulate “stress-response genes” in reaction to particular stressors. Comparative studies on diverse organisms have identified a number of evolutionarily conserved genes involved in responding to abiotic and biotic stressors. We used homology-based scans to survey the sequenced genome of Nematostella vectensis, the starlet sea anemone, an estuarine specialist, to identify genes involved in the response to three kinds of insult—physiochemical insults, pathogens, and injury. Many components of the stress-response networks identified in triploblastic animals have clear orthologs in the sea anemone, meaning that they must predate the cnidarian-triploblast split (e.g., xenobiotic receptors, biotransformative genes, ATP-dependent transporters, and genes involved in responding to reactive oxygen species, toxic metals, osmotic shock, thermal stress, pathogen exposure, and wounding). However, in some instances, stress-response genes known from triploblasts appear to be absent from the Nematostella genome (e.g., many metal-complexing genes). This is the first comprehensive examination of the genomic stress-response repertoire of an estuarine animal and a member of the phylum Cnidaria. The molecular markers of stress response identified in Nematostella may prove useful in monitoring estuary health and evaluating coastal conservation efforts. These data may also inform conservation efforts on other cnidarians, such as the reef-building corals.

Concerted evolution of sea anemone neurotoxin genes is revealed through analysis of the Nematostella vectensis genome.

Gene families, which encode toxins, are found in many poisonous animals, yet there is limited understanding of their evolution at the nucleotide level. The release of the genome draft sequence for the sea anemone Nematostella vectensis enabled a comprehensive study of a gene family whose neurotoxin products affect voltage-gated sodium channels. All gene family members are clustered in a highly repetitive approximately 30-kb genomic region and encode a single toxin, Nv1. These genes exhibit extreme conservation at the nucleotide level which cannot be explained by purifying selection. This conservation greatly differs from the toxin gene families of other animals (e.g., snakes, scorpions, and cone snails), whose evolution was driven by diversifying selection, thereby generating a high degree of genetic diversity. The low nucleotide diversity at the Nv1 genes is reminiscent of that reported for DNA encoding ribosomal RNA (rDNA) and 2 hsp70 genes from Drosophila, which have evolved via concerted evolution. This evolutionary pattern was experimentally demonstrated in yeast rDNA and was shown to involve unequal crossing-over. Through sequence analysis of toxin genes from multiple N. vectensis populations and 2 other anemone species, Anemonia viridis and Actinia equina, we observed that the toxin genes for each sea anemone species are more similar to one another than to those of other species, suggesting they evolved by manner of concerted evolution. Furthermore, in 2 of the species (A. viridis and A. equina) we found genes that evolved under diversifying selection, suggesting that concerted evolution and accelerated evolution may occur simultaneously.

Two Alleles of NF-κB in the Sea Anemone Nematostella vectensis Are Widely Dispersed in Nature and Encode Proteins with Distinct Activities

Background NF-κB is an evolutionarily conserved transcription factor that controls the expression of genes involved in many key organismal processes, including innate immunity, development, and stress responses. NF-κB proteins contain a highly conserved DNA-binding/dimerization domain called the Rel homology domain. Methods/Principal Findings We characterized two NF-κB alleles in the sea anemone Nematostella vectensis that differ at nineteen single-nucleotide polymorphisms (SNPs). Ten of these SNPs result in amino acid substitutions, including six within the Rel homology domain. Both alleles are found in natural populations of Nematostella. The relative abundance of the two NF-κB alleles differs between populations, and departures from Hardy-Weinberg equilibrium within populations indicate that the locus may be under selection. The proteins encoded by the two Nv-NF-κB alleles have different molecular properties, in part due to a Cys/Ser polymorphism at residue 67, which resides within the DNA recognition loop. In nearly all previously characterized NF-κB proteins, the analogous residue is fixed for Cys, and conversion of human RHD proteins from Cys to Ser at this site has been shown to increase DNA-binding ability and increase resistance to inhibition by thiol-reactive compounds. However, the naturally-occurring Nematostella variant with Cys at position 67 binds DNA with a higher affinity than the Ser variant. On the other hand, the Ser variant activates transcription in reporter gene assays more effectively, and it is more resistant to inhibition by a thiol-reactive compound. Reciprocal Cys<->Ser mutations at residue 67 of the native Nv-NF-κB proteins affect DNA binding as in human NF-κB proteins, e.g., a Cys->Ser mutation increases DNA binding of the native Cys variant. Conclusions/Significance These results are the first demonstration of a naturally occurring and functionally significant polymorphism in NF-κB in any species. The functional differences between these alleles and their uneven distribution in the wild suggest that different genotypes could be favored in different environments, perhaps environments that vary in their levels of peroxides or thiol-reactive compounds.

The evolutionary origin of the Runx/CBFbeta transcription factors – Studies of the most basal metazoans

BackgroundMembers of the Runx family of transcriptional regulators, which bind DNA as heterodimers with CBFβ, are known to play critical roles in embryonic development in many triploblastic animals such as mammals and insects. They are known to regulate basic developmental processes such as cell fate determination and cellular potency in multiple stem-cell types, including the sensory nerve cell progenitors of ganglia in mammals.ResultsIn this study, we detect and characterize the hitherto unexplored Runx/CBFβ genes of cnidarians and sponges, two basal animal lineages that are well known for their extensive regenerative capacity. Comparative structural modeling indicates that the Runx-CBFβ-DNA complex from most cnidarians and sponges is highly similar to that found in humans, with changes in the residues involved in Runx-CBFβ dimerization in either of the proteins mirrored by compensatory changes in the binding partner. In situ hybridization studies reveal that Nematostella Runx and CBFβ are expressed predominantly in small isolated foci at the base of the ectoderm of the tentacles in adult animals, possibly representing neurons or their progenitors.ConclusionThese results reveal that Runx and CBFβ likely functioned together to regulate transcription in the common ancestor of all metazoans, and the structure of the Runx-CBFβ-DNA complex has remained extremely conserved since the human-sponge divergence. The expression data suggest a hypothesis that these genes may have played a role in nerve cell differentiation or maintenance in the common ancestor of cnidarians and bilaterians.

Models developed from δ13C and δ15N of skin tissue indicate non-specific habitat use by the big brown bat (Eptesicus fuscus)

Abstract Stable isotopes can be used to evaluate trophic relationships, nutrient state, and temporal and spatial variation in diet, food webs, and behaviour both within and between species. Here we describe the development and application of models to predict habitat use of a common insectivorous bat (Eptesicus fuscus) based upon δ13C and δ15N signatures of skin tissue. We used a 42-specimen sample collected from three well-characterized ecogeographic regions, disparate both in photosynthetic mechanism and fertilizer use, to generate the models. Significant univariate differences between these three sites in terms of δ13C (F2, 39 = 112.92, P < 0.0001) and δ15N (F2, 39 = 97.06, P < 0.0001), and multivariate significance of both variables (Wilks λ = 0.032, F4, 76 = 87.02, P < 0.0001), made it possible to develop three predictive models using Fishers linear discriminant functions: 1) a model predicting if bats forage in C3 or mixed C3/C4 sites, 2) a model predicting if bats forage in agricultural areas, and 3) a combined model using both variables to predict specific habitat use. We present the results of model application to an independent dataset of 329 bats sampled from 10 states that included a broad range of δ13C (−26.53‰ δ13C −17.20‰) and δ15N (6.36‰ δ15N 15.60‰) signatures. We validated the use of skin tissue samples (from wing membranes) in the model by comparing the sites used for model development across five tissue types, selecting skin samples for model development due to consistently low variance within this tissue type. Our results indicate non-specific habitat-use by big brown bats.

Intron Retention as a Posttranscriptional Regulatory Mechanism of Neurotoxin Expression at Early Life Stages of the Starlet Anemone Nematostella vectensis

Sea anemones use an arsenal of peptide neurotoxins accumulated in special stinging cells (nematocytes) for defense and predation. Intriguingly, genomic analysis of Nematostella vectensis revealed only a single toxin, Nv1 (N. vectensis toxin 1), encoded by multiple extremely conserved genes. We examined the toxic potential of Nv1 and whether it is produced by the three developmental stages (embryo, planula, and polyp) of Nematostella. Nv1 was expressed in recombinant form and, similarly to Type I sea anemone toxins, inhibited the inactivation of voltage-gated sodium channels. However, in contrast to the other toxins, Nv1 revealed high specificity for insect over mammalian voltage-gated sodium channels. Transcript analysis indicated that multiple Nv1 loci are transcribed at all developmental stages of N. vectensis, whereas splicing of these transcripts is restricted to the polyp stage. This finding suggests that regulation of Nv1 synthesis is posttranscriptional and that the embryo and planula stages do not produce the Nv1 toxin. This rare phenomenon of intron retention at the early developmental stages is intriguing and raises the question as to the mechanism enabling such differential expression in sea anemones.

A surprising abundance of human disease genes in a simple "basal" animal, the starlet sea anemone (Nematostella vectensis).

Invertebrate animals have provided important insights into the mechanisms of, and treatment for, numerous human diseases. A surprisingly high proportion of genes underlying human disease are present in the genome of a simple, evolutionarily basal invertebrate animal, Nematostella vectensis, including some genes that are absent in established invertebrate model organisms. This, together with the laboratory tractability and regenerative capability of N. vectensis, recommends the species as an important new experimental model for the study of genes underlying human disease.

Upgrades to StellaBase facilitate medical and genetic studies on the starlet sea anemone, Nematostella vectensis

The starlet sea anemone, Nematostella vectensis, is a basal metazoan organism that has recently emerged as an important model system in developmental biology and evolutionary genomics. StellaBase, the Nematostella Genomics Database (http://stellabase.org), was developed in 2005 as a resource to support the Nematostella research community. Recently, it has become apparent that Nematostella may be a particularly useful system for studying (i) microevolutionary variation in natural populations, and (ii) the functional evolution of human disease genes. We have developed two new databases that will foster such studies: StellaBase Disease (http://stellabase.org/disease) is a relational database that houses 155 904 invertebrate homologous isoforms of human disease genes from four leading genomic model systems (fly, worm, yeast and Nematostella), including 14 874 predicted genes from the sea anemone itself. StellaBase SNP (http://stellabase.org/SNP) is a relational database that describes the location and underlying type of mutation for 20 063 single nucleotide polymorphisms.