ABSTRACT
In animals, stem cell populations of varying potency facilitate regeneration and tissue homeostasis. Notably, germline stem cells in both vertebrates and invertebrates express highly conserved RNA binding proteins, such as nanos, vasa, and piwi. In highly regenerative animals, these genes are also expressed in somatic stem cells, which led to the proposal that they had an ancestral role in all stem cells. In cnidarians, multi- and pluripotent interstitial stem cells have only been identified in hydrozoans. Therefore, it is currently unclear if cnidarian stem cell systems share a common evolutionary origin. We, therefore, aimed to characterize conserved stem cell marker genes in the sea anemone Nematostella vectensis. Through transgenic reporter genes and single-cell transcriptomics, we identify cell populations expressing the germline-associated markers piwi1 and nanos2 in the soma and germline, and gene knockout shows that Nanos2 is indispensable for germline formation. This suggests that nanos and piwi genes have a conserved role in somatic and germline stem cells in cnidarians.
Subject(s)
Germ Cells , RNA-Binding Proteins , Sea Anemones , Animals , Sea Anemones/genetics , Sea Anemones/metabolism , Germ Cells/metabolism , Germ Cells/cytology , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/genetics , Cell Lineage/genetics , Stem Cells/metabolism , Stem Cells/cytology , Argonaute Proteins/metabolism , Argonaute Proteins/geneticsABSTRACT
BACKGROUND: The starlet sea anemone, Nematostella vectensis, is an emerging model organism with a high regenerative capacity, which was recently found to possess an orthologue to the human Leucine Rich Repeat Kinase 2 (LRRK2) gene. Mutations in this gene are the most common cause of inherited Parkinson's Disease (PD), highlighting the importance of understanding its function. Despite two decades of research, however, the function of LRRK2 is not well established. METHODS: To investigate the function of LRRKs in Nematostella vectensis, we applied small molecule inhibitors targeting the kinase activity of LRRK2 to examine its function in development, homeostasis and regeneration in Nematostella vectensis. RESULTS: In vivo analyses inhibiting the kinase function of this enzyme demonstrated a role of nvLRRK2 in development and regeneration of N. vectensis. These findings implicate a developmental role of LRRK2 in Nematostella, adding to the expanding knowledge of its physiological function. CONCLUSIONS: Our work introduces a new model organism with which to study LRRK biology. We report that LRRK kinase activity is necessary for the development and regeneration of Nematostella. Given the short generation time, genetic trackability and in vivo imaging capabilities, this work introduces Nematostella vectensis as a new model in which to study genes linked to neurodegenerative diseases such as Parkinson's.
Subject(s)
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Sea Anemones , Animals , Sea Anemones/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/genetics , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolism , Regeneration/physiologyABSTRACT
The evolutionary transition from diffusion-mediated cell-cell communication to faster, targeted synaptic signaling in animal nervous systems is still unclear. Genome sequencing analyses have revealed a widespread distribution of synapse-related genes among early-diverging metazoans, but how synaptic machinery evolved remains largely unknown. Here, we examine the function of neurexins (Nrxns), a family of presynaptic cell adhesion molecules with critical roles in bilaterian chemical synapses, using the cnidarian model, Nematostella vectensis. Delta-Nrxns are expressed mainly in neuronal cell clusters that exhibit both peptidergic and classical neurotransmitter signaling. Knockdown of δ-Nrxn reduces spontaneous peristalsis of N. vectensis polyps. Interestingly, gene knockdown and pharmacological studies suggest that δ-Nrxn is involved in glutamate- and glycine-mediated signaling rather than peptidergic signaling. Knockdown of the epithelial α-Nrxn reveals a major role in cell adhesion between ectodermal and endodermal epithelia. Overall, this study provides molecular, functional, and cellular insights into the pre-neural function of Nrxns, as well as key information for understanding how and why they were recruited to the synaptic machinery.
Subject(s)
Neurexins , Neurons , Sea Anemones , Animals , Cell Adhesion/genetics , Gene Knockdown Techniques , Glutamic Acid/metabolism , Glycine/metabolism , Neural Cell Adhesion Molecules/metabolism , Neural Cell Adhesion Molecules/genetics , Neurons/metabolism , Sea Anemones/genetics , Sea Anemones/metabolism , Signal Transduction , Synapses/metabolism , Neurexins/metabolismABSTRACT
Sphingomyelin (SM) is a major sphingolipid in mammalian cells. SM is enriched in the extracellular leaflet of the plasma membrane (PM). Besides this localization, recent electron microscopic and biochemical studies suggest the presence of SM in the cytosolic leaflet of the PM. In the present study, we generated a non-toxic SM-binding variant (NT-EqtII) based on equinatoxin-II (EqtII) from the sea anemone Actinia equina, and examined the dynamics of SM in the cytosolic leaflet of living cell PMs. NT-EqtII with two point mutations (Leu26Ala and Pro81Ala) had essentially the same specificity and affinity to SM as wild-type EqtII. NT-EqtII expressed in the cytosol was recruited to the PM in various cell lines. Super-resolution microscopic observation revealed that NT-EqtII formed tiny domains that were significantly colocalized with cholesterol and N-terminal Lyn. Meanwhile, single molecule observation at high resolutions down to 1 ms revealed that all the examined lipid probes including NT-EqtII underwent apparent fast simple Brownian diffusion, exhibiting that SM and other lipids in the cytosolic leaflet rapidly moved in and out of domains. Thus, the novel SM-binding probe demonstrated the presence of the raft-like domain in the cytosolic leaflet of living cell PMs.
Subject(s)
Cell Membrane , Cnidarian Venoms , Cytosol , Sphingomyelins , Sphingomyelins/metabolism , Cell Membrane/metabolism , Cytosol/metabolism , Animals , Cnidarian Venoms/metabolism , Cnidarian Venoms/genetics , Humans , Sea Anemones/metabolism , Sea Anemones/genetics , Cholesterol/metabolismABSTRACT
Sea anemones are venomous animals that rely on their venom for prey capture, defense against predators, and intraspecific competition. Currently, comprehensive molecular and evolutionary analyses of the toxin repertoire for sea anemones are limited by a lack of proteomic data for most species. In this study, proteo-transcriptomic analysis was used to expand our knowledge of the proteinaceous components of sea anemone venom by determining the secreted venom proteome of Calliactis polypus. Electromechanical stimulation was used to obtain the secreted venom of C. polypus. We identified a low complexity proteome that was dominated by toxins with similarity to known neurotoxins, as well as six novel toxin candidates. The novel putative toxin candidates were found to be taxonomically restricted to species from the superfamily Metridioidea. Furthermore, the secreted venom of C. polypus had only three putative toxins in common with the venom of acontia from the same species and little similarity with the secreted venom of closely related species. Overall, this demonstrates that regionalized and lineage-specific variability in toxin abundance is common among sea anemone species. Moreover, the limited complexity of the toxin repertoire found in C. polypus supports the idea that peptide neurotoxins make up the dominant toxin arsenal found in the venom of sea anemones.
Subject(s)
Proteome , Sea Anemones , Animals , Sea Anemones/genetics , Cnidarian Venoms/genetics , Cnidarian Venoms/chemistry , Cnidarian Venoms/metabolism , Neurotoxins/genetics , Phylogeny , ProteomicsABSTRACT
Apoptosis, a major form of programmed cell death, is an essential component of host defense against invading intracellular pathogens. Viruses encode inhibitors of apoptosis to evade host responses during infection, and to support their own replication and survival. Therefore, hosts and their viruses are entangled in a constant evolutionary arms race to control apoptosis. Until now, apoptosis in the context of the antiviral immune system has been almost exclusively studied in vertebrates. This limited phyletic sampling makes it impossible to determine whether a similar mechanism existed in the last common ancestor of animals. Here, we established assays to probe apoptosis in the sea anemone Nematostella vectensis, a model species of Cnidaria, a phylum that diverged approximately 600 million years ago from the rest of animals. We show that polyinosinic:polycytidylic acid (poly I:C), a synthetic long double-stranded RNA mimicking viral RNA and a primary ligand for the vertebrate RLR melanoma differentiation-associated protein 5 (MDA5), is sufficient to induce apoptosis in N. vectensis. Furthermore, at the transcriptomic level, apoptosis related genes are significantly enriched upon poly(I:C) exposure in N. vectensis as well as bilaterian invertebrates. Our phylogenetic analysis of caspase family genes in N. vectensis reveals conservation of all four caspase genes involved in apoptosis in mammals and revealed a cnidarian-specific caspase gene which was strongly upregulated. Altogether, our findings suggest that apoptosis in response to a viral challenge is a functionally conserved mechanism that can be traced back to the last common ancestor of Bilateria and Cnidaria.
Subject(s)
Apoptosis , RNA, Double-Stranded , Sea Anemones , Animals , Sea Anemones/genetics , RNA, Double-Stranded/metabolism , RNA, Double-Stranded/genetics , Phylogeny , Poly I-C/pharmacology , Cnidaria/genetics , Biological EvolutionABSTRACT
Sea anemones (Order Actiniaria) are a diverse group of marine invertebrates ubiquitous across marine ecosystems. Despite their wide distribution and success, a knowledge gap persists in our understanding of their diversity within tropical systems, owed to sampling bias of larger and more charismatic species overshadowing cryptic lineages. This study aims to delineate the sea anemone diversity in Mo'orea (French Polynesia) with the use of a dataset from the Mo'orea Biocode's "BioBlitz" initiative, which prioritized the sampling of more cryptic and understudied taxa. Implementing a target enrichment approach, we integrate 71 newly sequenced samples into an expansive phylogenetic framework and contextualize Mo'orea's diversity within global distribution patterns of sea anemones. Our analysis corroborates the presence of several previously documented sea anemones in French Polynesia and identifies for the first time the occurrence of members of genera Andvakia and Aiptasiomorpha. This research unveils the diverse sea anemone ecosystem in Mo'orea, spotlighting the area's ecological significance and emphasizing the need for continued exploration. Our methodology, encompassing a broad BLAST search coupled with phylogenetic analysis, proved to be a practical and effective approach for overcoming the limitations posed by the lack of comprehensive sequence data for sea anemones. We discuss the merits and limitations of current molecular methodologies and stress the importance of further research into lesser-studied marine organisms like sea anemones. Our work sets a precedent for future phylogenetic studies stemming from BioBlitz endeavors.
Subject(s)
Phylogeny , Sea Anemones , Animals , Polynesia , Sea Anemones/genetics , Sea Anemones/classification , Biodiversity , Sequence Analysis, DNAABSTRACT
The complex morphology of neurons requires precise control of their microtubule cytoskeleton. This is achieved by microtubule-associated proteins (MAPs) that regulate the assembly and stability of microtubules, and transport of molecules and vesicles along them. While many of these MAPs function in all cells, some are specifically or predominantly involved in regulating microtubules in neurons. Here we use the sea anemone Nematostella vectensis as a model organism to provide new insights into the early evolution of neural microtubule regulation. As a cnidarian, Nematostella belongs to an outgroup to all bilaterians and thus occupies an informative phylogenetic position for reconstructing the evolution of nervous system development. We identified an ortholog of the microtubule-binding protein doublecortin-like kinase (NvDclk1) as a gene that is predominantly expressed in neurons and cnidocytes (stinging cells), two classes of cells belonging to the neural lineage in cnidarians. A transgenic NvDclk1 reporter line revealed an elaborate network of neurite-like processes emerging from cnidocytes in the tentacles and the body column. A transgene expressing NvDclk1 under the control of the NvDclk1 promoter suggests that NvDclk1 localizes to microtubules and therefore likely functions as a microtubule-binding protein. Further, we generated a mutant for NvDclk1 using CRISPR/Cas9 and show that the mutants fail to generate mature cnidocytes. Our results support the hypothesis that the elaboration of programs for microtubule regulation occurred early in the evolution of nervous systems.
Subject(s)
Doublecortin-Like Kinases , Neurons , Sea Anemones , Animals , Sea Anemones/embryology , Sea Anemones/cytology , Sea Anemones/genetics , Neurons/metabolism , Neurons/cytology , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Microtubules/metabolism , Neurogenesis/physiology , Animals, Genetically Modified , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/geneticsABSTRACT
Background: Antibiotics are commonly used for controlling microbial growth in diseased organisms. However, antibiotic treatments during early developmental stages can have negative impacts on development and physiology that could offset the positive effects of reducing or eliminating pathogens. Similarly, antibiotics can shift the microbial community due to differential effectiveness on resistant and susceptible bacteria. Though antibiotic application does not typically result in mortality of marine invertebrates, little is known about the developmental and transcriptional effects. These sublethal effects could reduce the fitness of the host organism and lead to negative changes after removal of the antibiotics. Here, we quantify the impact of antibiotic treatment on development, gene expression, and the culturable bacterial community of a model cnidarian, Nematostella vectensis. Methods: Ampicillin, streptomycin, rifampicin, and neomycin were compared individually at two concentrations, 50 and 200 µg mL-1, and in combination at 50 µg mL-1 each, to assess their impact on N. vectensis. First, we determined the impact antibiotics have on larval development. Next Amplicon 16S rDNA gene sequencing was used to compare the culturable bacteria that persist after antibiotic treatment to determine how these treatments may differentially select against the native microbiome. Lastly, we determined how acute (3-day) and chronic (8-day) antibiotic treatments impact gene expression of adult anemones. Results: Under most exposures, the time of larval settlement extended as the concentration of antibiotics increased and had the longest delay of 3 days in the combination treatment. Culturable bacteria persisted through a majority of exposures where we identified 359 amplicon sequence variants (ASVs). The largest proportion of bacteria belonged to Gammaproteobacteria, and the most common ASVs were identified as Microbacterium and Vibrio. The acute antibiotic exposure resulted in differential expression of genes related to epigenetic mechanisms and neural processes, while constant application resulted in upregulation of chaperones and downregulation of mitochondrial genes when compared to controls. Gene Ontology analyses identified overall depletion of terms related to development and metabolism in both antibiotic treatments. Discussion: Antibiotics resulted in a significant increase to settlement time of N. vectensis larvae. Culturable bacterial species after antibiotic treatments were taxonomically diverse. Additionally, the transcriptional effects of antibiotics, and after their removal result in significant differences in gene expression that may impact the physiology of the anemone, which may include removal of bacterial signaling on anemone gene expression. Our research suggests that impacts of antibiotics beyond the reduction of bacteria may be important to consider when they are applied to aquatic invertebrates including reef building corals.
Subject(s)
Anti-Bacterial Agents , Larva , Sea Anemones , Animals , Anti-Bacterial Agents/pharmacology , Sea Anemones/genetics , Sea Anemones/drug effects , Larva/microbiology , Larva/drug effects , Larva/genetics , Ampicillin/pharmacology , Neomycin/pharmacology , Streptomycin/pharmacology , Rifampin/pharmacology , Gene Expression/drug effectsABSTRACT
Neuropeptides are ancient signaling molecules in animals but only few peptide receptors are known outside bilaterians. Cnidarians possess a large number of G protein-coupled receptors (GPCRs) - the most common receptors of bilaterian neuropeptides - but most of these remain orphan with no known ligands. We searched for neuropeptides in the sea anemone Nematostella vectensis and created a library of 64 peptides derived from 33 precursors. In a large-scale pharmacological screen with these peptides and 161 N. vectensis GPCRs, we identified 31 receptors specifically activated by 1 to 3 of 14 peptides. Mapping GPCR and neuropeptide expression to single-cell sequencing data revealed how cnidarian tissues are extensively connected by multilayer peptidergic networks. Phylogenetic analysis identified no direct orthology to bilaterian peptidergic systems and supports the independent expansion of neuropeptide signaling in cnidarians from a few ancestral peptide-receptor pairs.
Subject(s)
Neuropeptides , Phylogeny , Receptors, G-Protein-Coupled , Sea Anemones , Animals , Sea Anemones/genetics , Neuropeptides/metabolism , Neuropeptides/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/genetics , Signal TransductionABSTRACT
The circadian clock enables anticipation of the day/night cycle in animals ranging from cnidarians to mammals. Circadian rhythms are generated through a transcription-translation feedback loop (TTFL or pacemaker) with CLOCK as a conserved positive factor in animals. However, CLOCK's functional evolutionary origin and mechanism of action in basal animals are unknown. In the cnidarian Nematostella vectensis, pacemaker gene transcript levels, including NvClk (the Clock ortholog), appear arrhythmic under constant darkness, questioning the role of NvCLK. Utilizing CRISPR/Cas9, we generated a NvClk allele mutant (NvClkΔ), revealing circadian behavior loss under constant dark (DD) or light (LL), while maintaining a 24 hr rhythm under light-dark condition (LD). Transcriptomics analysis revealed distinct rhythmic genes in wild-type (WT) polypsunder LD compared to DD conditions. In LD, NvClkΔ/Δ polyps exhibited comparable numbers of rhythmic genes, but were reduced in DD. Furthermore, under LD, the NvClkΔ/Δ polyps showed alterations in temporal pacemaker gene expression, impacting their potential interactions. Additionally, differential expression of non-rhythmic genes associated with cell division and neuronal differentiation was observed. These findings revealed that a light-responsive pathway can partially compensate for circadian clock disruption, and that the Clock gene has evolved in cnidarians to synchronize rhythmic physiology and behavior with the diel rhythm of the earth's biosphere.
Subject(s)
Circadian Clocks , Circadian Rhythm , Animals , Circadian Rhythm/genetics , Circadian Clocks/genetics , Sea Anemones/genetics , Sea Anemones/physiology , CLOCK Proteins/genetics , CLOCK Proteins/metabolism , Photoperiod , Cnidaria/physiology , Cnidaria/geneticsABSTRACT
Gene duplication is a major force driving evolutionary innovation. A classic example is generating new animal toxins via duplication of physiological protein-encoding genes and recruitment into venom. While this process drives the innovation of many animal venoms, reverse recruitment of toxins into nonvenomous cells remains unresolved. Using comparative genomics, we find members of the Membrane Attack Complex and Perforin Family (MAC) have been recruited into venom-injecting cells (cnidocytes), in soft and stony corals and sea anemones, suggesting that the ancestral MAC was a cnidocyte expressed toxin. Further investigation into the model sea anemone Nematostella vectensis reveals that three members have undergone Nematostella-specific duplications leading to their reverse recruitment into endomesodermal cells. Furthermore, simultaneous knockdown of all three endomesodermally expressed MACs leads to mis-development, supporting that these paralogs have nonvenomous function. By resolving the evolutionary history and function of MACs in Nematostella, we provide the first proof for reverse recruitment from venom to organismal development.
Subject(s)
Evolution, Molecular , Perforin , Sea Anemones , Animals , Sea Anemones/genetics , Perforin/metabolism , Perforin/genetics , Gene Duplication , Cnidarian Venoms/genetics , Cnidarian Venoms/metabolism , Phylogeny , Multigene FamilyABSTRACT
Bilaterian animals have evolved complex sensory organs comprised of distinct cell types that function coordinately to sense the environment. Each sensory unit has a defined architecture built from component cell types, including sensory cells, non-sensory support cells, and dedicated sensory neurons. Whether this characteristic cellular composition is present in the sensory organs of non-bilaterian animals is unknown. Here, we interrogate the cell type composition and gene regulatory networks controlling development of the larval apical sensory organ in the sea anemone Nematostella vectensis. Using single cell RNA sequencing and imaging approaches, we reveal two unique cell types in the Nematostella apical sensory organ, GABAergic sensory cells and a putative non-sensory support cell population. Further, we identify the paired-like (PRD) homeodomain gene prd146 as a specific sensory cell marker and show that Prd146+ sensory cells become post-mitotic after gastrulation. Genetic loss of function approaches show that Prd146 is essential for apical sensory organ development. Using a candidate gene knockdown approach, we place prd146 downstream of FGF signaling in the apical sensory organ gene regulatory network. Further, we demonstrate that an aboral FGF activity gradient coordinately regulates the specification of both sensory and support cells. Collectively, these experiments define the genetic basis for apical sensory organ development in a non-bilaterian animal and reveal an unanticipated degree of complexity in a prototypic sensory structure.
Subject(s)
Sea Anemones , Animals , Sea Anemones/genetics , Nervous System , Gastrulation/genetics , Genes, HomeoboxABSTRACT
The ability of an animal to effectively capture prey and defend against predators is pivotal for survival. Venom is often a mixture of many components including toxin proteins that shape predator-prey interactions. Here, we used the sea anemone Nematostella vectensis to test the impact of toxin genotypes on predator-prey interactions. We developed a genetic manipulation technique to demonstrate that both transgenically deficient and a native Nematostella strain lacking a major neurotoxin (Nv1) have a reduced ability to defend themselves against grass shrimp, a native predator. In addition, secreted Nv1 can act indirectly in defense by attracting mummichog fish, which prey on grass shrimp. Here, we provide evidence at the molecular level of an animal-specific tritrophic interaction between a prey, its antagonist, and a predator. Last, this study reveals an evolutionary trade-off, as the reduction of Nv1 levels allows for faster growth and increased reproductive rates.
Subject(s)
Sea Anemones , Venoms , Animals , Reproduction , Biological Evolution , Neurotoxins/genetics , Sea Anemones/genetics , Predatory Behavior/physiologyABSTRACT
Fluorescent proteins (FPs) are ubiquitous tools in research, yet their endogenous functions in nature are poorly understood. In this work, we describe a combination of functions for FPs in a clade of intertidal sea anemones whose FPs control a genetic color polymorphism together with the ability to combat oxidative stress. Focusing on the underlying genetics of a fluorescent green "Neon" color morph, we show that allelic differences in a single FP gene generate its strong and vibrant color, by increasing both molecular brightness and FP gene expression level. Natural variation in FP sequences also produces differences in antioxidant capacity. We demonstrate that these FPs are strong antioxidants that can protect live cells against oxidative stress. Finally, based on structural modeling of the responsible amino acids, we propose a model for FP antioxidant function that is driven by molecular surface charge. Together, our findings shed light on the multifaceted functions that can co-occur within a single FP and provide a framework for studying the evolution of fluorescence as it balances spectral and physiological functions in nature.
Subject(s)
Sea Anemones , Animals , Luminescent Proteins/metabolism , Sea Anemones/genetics , Sea Anemones/metabolism , Antioxidants/metabolism , Spectrometry, Fluorescence , Oxidative Stress/genetics , Green Fluorescent Proteins/metabolismABSTRACT
The sea anemone Nematostella vectensis is a genetically tractable cnidarian species that has become a model organism for studying the evolution of developmental processes and genome regulation, resilience to fluctuations in environmental conditions, and the response to pollutants. Gene expression analyses are central to many of these studies, and in situ hybridization has been an important method for obtaining spatial information, in particular during embryonic development. Like other cnidarians, Nematostella embryos are of comparably low morphological complexity, but they possess many cell types that are dispersed throughout the tissue and originate from broad and overlapping areas. These features have made two-color fluorescence in situ hybridization an important method to determine potential co-expression of genes and to generate hypotheses for their functions in cell fate specification. We here share protocols for single and double fluorescence in situ hybridization in Nematostella and for the combination of fluorescence in situ hybridization and immunofluorescence.
Subject(s)
Sea Anemones , Animals , Sea Anemones/genetics , In Situ Hybridization, Fluorescence , Cell Differentiation/genetics , Embryonic DevelopmentABSTRACT
Efficient and accurate acquisition of cellular biomolecular information is crucial for exploring cell fate, achieving early diagnosis, and the effective treatment of various diseases. However, current DNA biosensors are mostly limited to single-target detection, with few complex logic circuits for comprehensive analysis of three or more targets. Herein, we designed a sea anemone-like DNA nanomachine based on DNA strand displacement composed of three logic gates (YES-AND-YES) and delivered into the cells using gold nano bipyramid carriers. The AND gate activation depends on the trigger chain released by upstream DNA strand displacement reactions, while the output signal relies on the downstream DNAzyme structure. Under the influence of diverse inputs (including enzymes, miRNA, and metal ions), the interconnected logic gates simultaneously perform logical analysis on multiple targets, generating a unique output signal in the YES/NO format. This sensor can successfully distinguish healthy cells from tumor cells and can be further used for the diagnosis of different tumor cells, providing a promising platform for accurate cell-type identification.
Subject(s)
DNA, Catalytic , Sea Anemones , Animals , Sea Anemones/genetics , DNA/chemistry , DNA, Catalytic/chemistry , Logic , Gold , Computers, MolecularABSTRACT
Cnidarians (corals, sea anemones, and jellyfish) produce toxins that play central roles in key ecological processes, including predation, defense, and competition, being the oldest extant venomous animal lineage. Cnidaria small cysteine-rich proteins (SCRiPs) were the first family of neurotoxins detected in stony corals, one of the ocean's most crucial foundation species. Yet, their molecular evolution remains poorly understood. Moreover, the lack of a clear classification system has hindered the establishment of an accurate and phylogenetically informed nomenclature. In this study, we extensively surveyed 117 genomes and 103 transcriptomes of cnidarians to identify orthologous SCRiP gene sequences. We annotated a total of 168 novel putative SCRiPs from over 36 species of stony corals and 12 species of sea anemones. Phylogenetic reconstruction identified four distinct SCRiP subfamilies, according to strict discrimination criteria based on well-supported monophyly with a high percentage of nucleotide and amino acids' identity. Although there is a high prevalence of purifying selection for most SCRiP subfamilies, with few positively selected sites detected, a subset of Acroporidae sequences is influenced by diversifying positive selection, suggesting potential neofunctionalizations related to the fine-tuning of toxin potency. We propose a new nomenclature classification system relying on the phylogenetic distribution and evolution of SCRiPs across Anthozoa, which will further assist future proteomic and functional research efforts.
Subject(s)
Anthozoa , Cnidaria , Sea Anemones , Animals , Anthozoa/genetics , Sea Anemones/genetics , Cnidaria/genetics , Neurotoxins/genetics , Cysteine/genetics , Phylogeny , ProteomicsABSTRACT
While the unique symbiotic relationship between anemonefishes and sea anemones is iconic, it is still not fully understood how anemonefishes can withstand and thrive within the venomous environment of their host sea anemone. In this study, we used a proteotranscriptomics approach to elucidate the proteinaceous toxin repertoire from the most common host sea anemone, Entacmaea quadricolor. Although 1251 different toxin or toxin-like RNA transcripts were expressed in E. quadricolor tentacles (0.05% of gene clusters, 1.8% of expression) and 5375 proteins were detected in milked venom, only 4% of proteins detected in venom were putative toxins (230), and they only represent on average 14% of the normalised protein expression in the milked venom samples. Thus, most proteins in milked venom do not appear to have a toxin function. This work raises the perils of defining a dominant venom phenotype based on transcriptomics data alone in sea anemones, as we found that the dominant venom phenotype differs between the transcriptome and proteome abundance data. E. quadricolor venom contains a mixture of toxin-like proteins of unknown and known function. A newly identified toxin protein family, Z3, rich in conserved cysteines of unknown function, was the most abundant at the RNA transcript and protein levels. The venom was also rich in toxins from the Protease S1, Kunitz-type and PLA2 toxin protein families and contains toxins from eight venom categories. Exploring the intricate venom toxin components in other host sea anemones will be crucial for improving our understanding of how anemonefish adapt to the venomous environment.
Subject(s)
Sea Anemones , Toxins, Biological , Animals , Sea Anemones/genetics , Venoms/genetics , Toxins, Biological/genetics , Transcriptome , RNAABSTRACT
BMP signaling has a conserved function in patterning the dorsal-ventral body axis in Bilateria and the directive axis in anthozoan cnidarians. So far, cnidarian studies have focused on the role of different BMP signaling network components in regulating pSMAD1/5 gradient formation. Much less is known about the target genes downstream of BMP signaling. To address this, we generated a genome-wide list of direct pSMAD1/5 target genes in the anthozoan Nematostella vectensis, several of which were conserved in Drosophila and Xenopus. Our ChIP-seq analysis revealed that many of the regulatory molecules with documented bilaterally symmetric expression in Nematostella are directly controlled by BMP signaling. We identified several so far uncharacterized BMP-dependent transcription factors and signaling molecules, whose bilaterally symmetric expression may be indicative of their involvement in secondary axis patterning. One of these molecules is zswim4-6, which encodes a novel nuclear protein that can modulate the pSMAD1/5 gradient and potentially promote BMP-dependent gene repression.