Sea Anemone Nematostella Vectensis Research Articles

Mesoscopic axially swept oblique plane microscope for the imaging of freely moving organisms with near-isotropic resolution

Rapid three-dimensional imaging over extended fields of view (FOVs) is crucial to the study of organism-wide systems and biological processes in vivo. Selective-plane illumination microscopy (SPIM) is a powerful method for high spatio-temporal resolution in toto imaging of such biological specimens. However, typical SPIM implementations preclude conventional sample mounting and have anisotropic imaging performance, in particular when designed for large FOVs over 1 mm diameter. Here, we introduce axial sweeping of the illumination into a non-orthogonal dual-objective oblique plane microscope (OPM) design, thereby enabling the observation of freely moving animals over millimeter-sized FOVs, at close to isotropic, sub-cellular resolution. We apply our mesoscopic axially swept OPM (MASOPM) to image the behavioral dynamics of the sea anemone Nematostella vectensis over 1 × 0.7 × 0.4 mm at 1.7 × 2.6 × 3.7 µm resolution and 0.5 Hz volume rate.

Lateral cell polarization drives organization of epithelia in sea anemone embryos and embryonic cell aggregates

One of the first organizing processes during animal development is the assembly of embryonic cells into epithelia. Common features unite epithelialization across select bilaterians, however, we know less about the molecular and cellular mechanisms that drive epithelial emergence in early branching nonbilaterians. In sea anemones, epithelia emerge both during embryonic development and after cell aggregation of dissociated tissues. Although adhesion is required to keep cells together, it is not clear whether cell polarization plays a role as epithelia emerge from disordered aggregates. Here, we use the embryos of the sea anemone Nematostella vectensis to investigate the evolutionary origins of epithelial development. We demonstrate that lateral cell polarization is essential for epithelial organization in both embryos and aggregates. With disrupted lateral polarization, cell contact in the aggregate is not sufficient to trigger epithelialization and further tissue development. Specifically, knockdown of the conserved lateral polarity and tumor suppressor protein Lethal giant larvae (Lgl) disrupts epithelia in developing embryos and impairs the capacity of dissociated cells to epithelialize from aggregates. In contrast to other systems, cells in Nematostella lgl knockdown embryos do not undergo excessive proliferation. Cells with reduced Lgl levels lose their columnar shape and proper positioning of their mitotic spindles and basal bodies. Due to misoriented divisions and aberrant shapes, cells arrange nonuniformly without forming a monolayer. Together our data show that, in Nematostella, Lgl drives epithelialization in embryos and cell aggregates through its effect on cell shape and organelle localization.

A population of Vasa2 and Piwi1 expressing cells generates germ cells and neurons in a sea anemone

Germline segregation, essential for protecting germ cells against mutations, occurs during early embryogenesis in vertebrates, insects and nematodes. Highly regenerative animals (e.g., cnidarians), however, retain stem cells with both germinal and somatic potentials throughout adulthood, but their biology and evolution remain poorly understood. Among cnidarians (e.g., sea anemones, jellyfish), stem cells are only known in few hydrozoans (e.g., Hydra). Here, we identify and characterize a rare, multipotent population of stem and/or progenitor cells expressing the conserved germline and multipotency proteins Vasa2 and Piwi1 in the sea anemone Nematostella vectensis. Using piwi1 and vasa2 transgenic reporter lines, we reveal that the Vasa2+/Piwi1+ cell population generates not only gametes, but also a diversity of proliferative somatic cells, including neural progenitors, in juveniles and adults. Our work has uncovered a multipotent population of Vasa2+/Piwi1+ stem/progenitor cells that forms the cellular basis to understand body plasticity and regenerative capacities in sea anemones and corals.

Nanos2 marks precursors of somatic lineages and is required for germline formation in the sea anemone Nematostella vectensis.

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.

Induction of apoptosis by double-stranded RNA was present in the last common ancestor of cnidarian and bilaterian animals.

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.

Doublecortin-like kinase is required for cnidocyte development in Nematostella vectensis

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.

Large-scale deorphanization of Nematostella vectensis neuropeptide G protein-coupled receptors supports the independent expansion of bilaterian and cnidarian peptidergic systems.

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.

Large-scale deorphanization of Nematostella vectensis neuropeptide G protein-coupled receptors supports the independent expansion of bilaterian and cnidarian peptidergic systems

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.

Sea Anemone Membrane Attack Complex/Perforin Superfamily Demonstrates an Evolutionary Transitional State between Venomous and Developmental Functions.

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.

Larval swimming in the sea anemone Nematostella vectensis is sensitive to a broad light spectrum and exhibits a wavelength-dependent behavioral switch.

In nearly all animals, light-sensing mediated by opsin visual pigments is important for survival and reproduction. Eyeless light-sensing systems, though vital for many animals, have received relatively less attention than forms with charismatic or complex eyes. Despite no single light-sensing organ, the sea anemone Nematostella vectensis has 29 opsin genes and multiple light-mediated behaviors throughout development and reproduction, suggesting a deceptively complex light-sensing system. To characterize one aspect of this light-sensing system, we analyzed larval swimming behavior at high wavelength resolution across the ultraviolet and visual spectrum. N. vectensis larvae respond to light at least from 315 to 650 nm, which is a broad sensitivity range even compared to many animals with complex eyes. Planktonic swimming is induced by ultraviolet (UV) and violet wavelengths until 420 nm. Between 420 and 430 nm a behavioral switch occurs where at wavelengths longer than 430 nm, larvae respond to light by swimming down. Swimming down toward the substrate is distinct from light avoidance, as animals do not exhibit positive or negative phototaxis at any wavelength tested. At wavelengths longer than 575 nm, animals in the water column take increasingly longer to respond and this behavior is more variable until 650 nm where larval response is no different from the dark, suggesting these longer wavelengths lie outside of their sensitivity range. Larval swimming is the only motile stage in the life history of N. vectensis, and increased planktonic swimming could lead to greater dispersal range in potentially damaging shallow environments with short-wavelength light exposure. Longer wavelength environments may indicate more suitable substrates for metamorphosis into the polyp stage, where the individual will remain for the rest of its life. Future work will test whether this robust behavior is mediated by multiple opsins.

Graded FGF activity patterns distinct cell types within the apical sensory organ of the sea anemone Nematostella vectensis

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.

Updated single cell reference atlas for the starlet anemone Nematostella vectensis.

The recent combination of genomics and single cell transcriptomics has allowed to assess a variety of non-conventional model organisms in much more depth. Single cell transcriptomes can uncover hidden cellular complexity and cell lineage relationships within organisms. The recent developmental cell atlases of the sea anemone Nematostella vectensis, a representative of the basally branching Cnidaria, has provided new insights into the development of all cell types (Steger et al Cell Rep 40(12):111370, 2022; Sebé-Pedrós et al. Cell 173(6):1520-1534.e20). However, the mapping of the single cell reads still suffers from relatively poor gene annotations and a draft genome consisting of many scaffolds. Here we present a new wildtype resource of the developmental single cell atlas, by re-mapping of sequence data first published in Steger et al. (2022) and Cole et al. (Nat Commun 14(1):1747, 2023), to the new chromosome-level genome assembly and corresponding gene models in Zimmermann et al. (Nat Commun 14, 8270 (2023). https://doi.org/10.1038/s41467-023-44080-7 ). We expand the pre-existing dataset through the incorporation of additional sequence data derived from the capture and sequencing of cell suspensions from four additional samples: 24h gastrula, 2d planula, an inter-parietal region of the bodywall from a young unsexed animal, and another adult mesentery from a mature male animal. Our analyses of the full cell-state inventory provide transcriptomic signatures for 127 distinct cell states, of which 47 correspond to neuroglandular subtypes. We also identify two distinct putatively immune-related transcriptomic profiles that segregate between the inner and outer cell layers. Furthermore, the new gene annotation Nv2 has markedly improved the mapping on the single cell transcriptome data and will therefore be of great value for the community and anyone using the dataset.

Venom trade-off shapes interspecific interactions, physiology, and reproduction.

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.

Fluorescence In Situ Hybridization as a Tool for Studying the Specification and Differentiation of Cell Types in Nematostella vectensis.

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.

Acute heat priming promotes short-term climate resilience of early life stages in a model sea anemone

Across diverse taxa, sublethal exposure to abiotic stressors early in life can lead to benefits such as increased stress tolerance upon repeat exposure. This phenomenon, known as hormetic priming, is largely unexplored in early life stages of marine invertebrates, which are increasingly threatened by anthropogenic climate change. To investigate this phenomenon, larvae of the sea anemone and model marine invertebrate Nematostella vectensis were exposed to control (18 °C) or elevated (24 °C, 30 °C, 35 °C, or 39 °C) temperatures for 1 h at 3 days post-fertilization (DPF), followed by return to control temperatures (18 °C). The animals were then assessed for growth, development, metabolic rates, and heat tolerance at 4, 7, and 11 DPF. Priming at intermediately elevated temperatures (24 °C, 30 °C, or 35 °C) augmented growth and development compared to controls or priming at 39 °C. Indeed, priming at 39 °C hampered developmental progression, with around 40% of larvae still in the planula stage at 11 DPF, in contrast to 0% for all other groups. Total protein content, a proxy for biomass, and respiration rates were not significantly affected by priming, suggesting metabolic resilience. Heat tolerance was quantified with acute heat stress exposures, and was significantly higher for animals primed at intermediate temperatures (24 °C, 30 °C, or 35 °C) compared to controls or those primed at 39 °C at all time points. To investigate a possible molecular mechanism for the observed changes in heat tolerance, the expression of heat shock protein 70 (HSP70) was quantified at 11 DPF. Expression of HSP70 significantly increased with increasing priming temperature, with the presence of a doublet band for larvae primed at 39 °C, suggesting persistent negative effects of priming on protein homeostasis. Interestingly, primed larvae in a second cohort cultured to 6 weeks post-fertilization continued to display hormetic growth responses, whereas benefits for heat tolerance were lost; in contrast, negative effects of short-term exposure to extreme heat stress (39 °C) persisted. These results demonstrate that some dose-dependent effects of priming waned over time while others persisted, resulting in heterogeneity in organismal performance across ontogeny following priming. Overall, these findings suggest that heat priming may augment the climate resilience of marine invertebrate early life stages via the modulation of key developmental and physiological phenotypes, while also affirming the need to limit further anthropogenic ocean warming.

An ancient pan-cnidarian microRNA regulates stinging capsule biogenesis in Nematostella vectensis

An ancient evolutionary innovation of a novel cell type, the stinging cell (cnidocyte), appeared >600 million years ago in the phylum Cnidaria (sea anemones, corals, hydroids, and jellyfish). A complex bursting nano-injector of venom, the cnidocyst, is embedded in cnidocytes and enables cnidarians to paralyze their prey and predators, contributing to this phylum's evolutionary success. In this work, we show that post-transcriptional regulation by a pan-cnidarian microRNA, miR-2022, is essential for biogenesis of these cells in the sea anemone Nematostella vectensis. By manipulation of miR-2022 levels in a transgenic reporter line of cnidocytes, followed by transcriptomics, single-cell data analysis, prey paralysis assays, and cell sorting of transgenic cnidocytes, we reveal that miR-2022 enables cnidocyte biogenesis in Nematostella, while exhibiting a conserved expression domain with its targets in cnidocytes of other cnidarian species. Thus, here we revealed a functional basis to the conservation of one of nature's most ancient microRNAs.

Starvation decreases immunity and immune regulatory factor NF-κB in the starlet sea anemone Nematostella vectensis

Lack of proper nutrition has important consequences for the physiology of all organisms, and nutritional status can affect immunity, based on many studies in terrestrial animals. Here we show a positive correlation between nutrition and immunity in the sea anemone Nematostella vectensis. Gene expression profiling of adult anemones shows downregulation of genes involved in nutrient metabolism, cellular respiration, and immunity in starved animals. Starved adult anemones also have reduced protein levels and activity of immunity transcription factor NF-κB. Starved juvenile anemones have increased sensitivity to bacterial infection and also have lower NF-κB protein levels, as compared to fed controls. Weighted Gene Correlation Network Analysis (WGCNA) is used to identify significantly correlated gene networks that were downregulated with starvation. These experiments demonstrate a correlation between nutrition and immunity in an early diverged marine metazoan, and the results have implications for the survival of marine organisms as they encounter changing environments.

Spatial transcriptomics reveals a cnidarian segment polarity program in Nematostella vectensis

During early animal evolution, the emergence of axially polarized segments was central to the diversification of complex bilaterian body plans. Nevertheless, precisely how and when segment polarity pathways arose remains obscure. Here, we demonstrate the molecular basis for segment polarization in developing larvae of the sea anemone Nematostella vectensis. Utilizing spatial transcriptomics, we first constructed a 3D gene expression atlas of developing larval segments. Capitalizing on accurate in silico predictions, we identified Lbx and Uncx, conserved homeodomain-containing genes that occupy opposing subsegmental domains under the control of both bone morphogenetic protein (BMP) signaling and the Hox-Gbx cascade. Functionally, Lbx mutagenesis eliminated all molecular evidence of segment polarization at the larval stage and caused an aberrant mirror-symmetric pattern of retractor muscles (RMs) in primary polyps. These results demonstrate the molecular basis for segment polarity in a non-bilaterian animal, suggesting that polarized metameric structures were present in the Cnidaria-Bilateria common ancestor over 600 million years ago.

Sensory conflict disrupts circadian rhythms in the sea anemone Nematostella vectensis.

Circadian clocks infer time of day by integrating information from cyclic environmental factors called zeitgebers, including light and temperature. Single zeitgebers entrain circadian rhythms, but few studies have addressed how multiple, simultaneous zeitgeber cycles interact to affect clock behavior. Misalignment between zeitgebers ('sensory conflict') can disrupt circadian rhythms, or alternatively clocks may privilege information from one zeitgeber over another. Here, we show that temperature cycles modulate circadian locomotor rhythms in Nematostella vectensis, a model system for cnidarian circadian biology. We conduct behavioral experiments across a comprehensive range of light and temperature cycles and find that Nematostella's circadian behavior is disrupted by chronic misalignment between light and temperature, which involves disruption of the endogenous clock itself rather than a simple masking effect. Sensory conflict also disrupts the rhythmic transcriptome, with numerous genes losing rhythmic expression. However, many metabolic genes remained rhythmic and in-phase with temperature, and other genes even gained rhythmicity, implying that some rhythmic metabolic processes persist even when behavior is disrupted. Our results show that a cnidarian clock relies on information from light and temperature, rather than prioritizing one signal over the other. Although we identify limits to the clock's ability to integrate conflicting sensory information, there is also a surprising robustness of behavioral and transcriptional rhythmicity.

Muscle cell-type diversification is driven by bHLH transcription factor expansion and extensive effector gene duplications

Animals are typically composed of hundreds of different cell types, yet mechanisms underlying the emergence of new cell types remain unclear. Here we address the origin and diversification of muscle cells in the non-bilaterian, diploblastic sea anemone Nematostella vectensis. We discern two fast and two slow-contracting muscle cell populations, which differ by extensive sets of paralogous structural protein genes. We find that the regulatory gene set of the slow cnidarian muscles is remarkably similar to the bilaterian cardiac muscle, while the two fast muscles differ substantially from each other in terms of transcription factor profiles, though driving the same set of structural protein genes and having similar physiological characteristics. We show that anthozoan-specific paralogs of Paraxis/Twist/Hand-related bHLH transcription factors are involved in the formation of fast and slow muscles. Our data suggest that the subsequent recruitment of an entire effector gene set from the inner cell layer into the neural ectoderm contributes to the evolution of a novel muscle cell type. Thus, we conclude that extensive transcription factor gene duplications and co-option of effector modules act as an evolutionary mechanism underlying cell type diversification during metazoan evolution.