Basal Metazoan Research Articles

Molecular and Biochemical Approaches to Study the Evolution of NF-κB Signaling in Basal Metazoans.

Extensive genomic and transcriptomic sequencing over the past decade has revealed NF-κB signaling pathway homologs in organisms basal to insects, for example, in members of the phyla Cnidaria (e.g., sea anemones, corals, hydra, jellyfish) and Porifera (sponges), and in several single-celled protists (e.g., Capsaspora owczarzaki, some choanoflagellates). Therefore, methods are required to study the function of NF-κB and its pathway members in early branching organisms, many of which do not have histories as model organisms. Here, we describe a combination of cellular, molecular, and biochemical techniques that have been used for studying NF-κB, and related pathway proteins, in some of these basal organisms. These methods are useful for studying the evolution of NF-κB signaling, and may be adaptable to the study of NF-κB in other non-model organisms.

Developmental Symbiosis: A Sponge Larva Needs Symbiotic Bacteria to Succeed on the Benthos

Settlement and metamorphosis of marine invertebrate larvae are processes of profound developmental, morphological, physiological and ecological change. That these processes in larvae of a marine sponge critically rely on products supplied by endosymbiotic bacteria signals the importance of developmental symbiosis among the most basal metazoans.

The Native Microbiome is Crucial for Offspring Generation and Fitness of Aurelia aurita.

All multicellular organisms are associated with microbial communities, ultimately forming a metaorganism. Several studies conducted on well-established model organisms point to immunological, metabolic, and behavioral benefits of the associated microbiota for the host. Consequently, a microbiome can influence the physiology of a host; moreover, microbial community shifts can affect host health and fitness. The present study aimed to evaluate the significance and functional role of the native microbiota for life cycle transitions and fitness of the cnidarian moon jellyfish Aurelia aurita A comprehensive host fitness experiment was conducted studying the polyp life stage and integrating 12 combinations of treatments with microbiota modification (sterile conditions, foreign food bacteria, and potential pathogens). Asexual reproduction, e.g., generation of daughter polyps, and the formation and release of ephyrae were highly affected in the absence of the native microbiota, ultimately resulting in a halt of strobilation and ephyra release. Assessment of further fitness traits showed that health, growth, and feeding rate were decreased in the absence and upon community changes of the native microbiota, e.g., when challenged with selected bacteria. Moreover, changes in microbial community patterns were detected by 16S rRNA amplicon sequencing during the course of the experiment. This demonstrated that six operational taxonomic units (OTUs) significantly correlated and explained up to 97% of fitness data variability, strongly supporting the association of impaired fitness with the absence/presence of specific bacteria. Conclusively, our study provides new insights into the importance and function of the microbiome for asexual reproduction, health, and fitness of the basal metazoan A. auritaIMPORTANCE All multicellular organisms are associated with a diverse and specific community of microorganisms; consequently, the microbiome is of fundamental importance for health and fitness of the multicellular host. However, studies on microbiome contribution to host fitness are in their infancy, in particular, for less well-established hosts such as the moon jellyfish Aurelia aurita Here, we studied the impact of the native microbiome on the asexual reproduction and on further fitness traits (health, growth, and feeding) of the basal metazoan due to induced changes in its microbiome. We observed significant impact on all fitness traits analyzed, in particular, in the absence of the protective microbial shield and when challenged with marine potentially pathogenic bacterial isolates. Notable is the identified crucial importance of the native microbiome for the generation of offspring, consequently affecting life cycle decisions. Thus, we conclude that the microbiome is essential for the maintenance of a healthy metaorganism.

Epigenomic landscape of enhancer elements during Hydra head organizer formation

BackgroundAxis patterning during development is accompanied by large-scale gene expression changes. These are brought about by changes in the histone modifications leading to dynamic alterations in chromatin architecture. The cis regulatory DNA elements also play an important role towards modulating gene expression in a context-dependent manner. Hydra belongs to the phylum Cnidaria where the first asymmetry in the body plan was observed and the oral-aboral axis originated. Wnt signaling has been shown to determine the head organizer function in the basal metazoan Hydra.ResultsTo gain insights into the evolution of cis regulatory elements and associated chromatin signatures, we ectopically activated the Wnt signaling pathway in Hydra and monitored the genome-wide alterations in key histone modifications. Motif analysis of putative intergenic enhancer elements from Hydra revealed the conservation of bilaterian cis regulatory elements that play critical roles in development. Differentially regulated enhancer elements were identified upon ectopic activation of Wnt signaling and found to regulate many head organizer specific genes. Enhancer activity of many of the identified cis regulatory elements was confirmed by luciferase reporter assay. Quantitative chromatin immunoprecipitation analysis upon activation of Wnt signaling further confirmed the enrichment of H3K27ac on the enhancer elements of Hv_Wnt5a, Hv_Wnt11 and head organizer genes Hv_Bra1, CnGsc and Hv_Pitx1. Additionally, perturbation of the putative H3K27me3 eraser activity using a specific inhibitor affected the ectopic activation of Wnt signaling indicating the importance of the dynamic changes in the H3K27 modifications towards regulation of the genes involved in the head organizer activity.ConclusionsThe activation-associated histone marks H3K4me3, H3K27ac and H3K9ac mark chromatin in a similar manner as seen in bilaterians. We identified intergenic cis regulatory elements which harbor sites for key transcription factors involved in developmental processes. Differentially regulated enhancers exhibited motifs for many zinc-finger, T-box and ETS related TFs whose homologs have a head specific expression in Hydra and could be a part of the pioneer TF network in the patterning of the head. The ability to differentially modify the H3K27 residue is critical for the patterning of Hydra axis revealing a dynamic acetylation/methylation switch to regulate gene expression and chromatin architecture.

Dynamic interactions within the host-associated microbiota cause tumor formation in the basal metazoan Hydra.

The extent to which disturbances in the resident microbiota can compromise an animal’s health is poorly understood. Hydra is one of the evolutionary oldest animals with naturally occurring tumors. Here, we found a causal relationship between an environmental spirochete (Turneriella spec.) and tumorigenesis in Hydra. Unexpectedly, virulence of this pathogen requires the presence of Pseudomonas spec., a member of Hydra´s beneficial microbiome indicating that dynamic interactions between a resident bacterium and a pathogen cause tumor formation. The observation points to the crucial role of commensal bacteria in maintaining tissue homeostasis and adds support to the view that microbial community interactions are essential for disease. These findings in an organism that shares deep evolutionary connections with all animals have implications for our understanding of cancer.

Intergenerational epigenetic inheritance in reef-building corals

The perception that the inheritance of phenotypic traits operates solely through genetic means is slowly being eroded: epigenetic mechanisms have been shown to induce heritable changes in gene activity in plants1,2 and metazoans1,3. Inheritance of DNA methylation patterns provides a potential pathway for environmentally induced phenotypes to contribute to evolution of species and populations1–5. However, in basal metazoans, it is unknown whether inheritance of CpG methylation patterns occurs across the genome (as in plants) or as rare exceptions (as in mammals)4. Here, we show that DNA methylation patterns in a reef-building coral are determined by genotype and developmental stage, as well as by parental environment. Transmission of CpG methylation from adults to their sperm and larvae demonstrates genome-wide inheritance. Variation in the hypermethylation of genes in adults and their sperm from distinct environments suggests intergenerational acclimatization to local temperature and salinity. Furthermore, genotype-independent adjustments of methylation levels in stress-related genes were strongly correlated with offspring survival rates under heat stress. These findings support a role of DNA methylation in the intergenerational inheritance of traits in corals, which could extend to enhancing their capacity to adapt to climate change. Intergenerational inheritance of traits in corals can help species survive environmental change. Examination of intergenerational DNA methylation profiles in a reef-building coral shows there to be genome-wide inheritance, with the potential for adaptive capacity to environmental stressors.

Differential expression of Exaiptasia pallida GIMAP genes upon induction of apoptosis and autophagy suggests a potential role in cnidarian symbiosis and disease.

Coral reefs, one of the world's most productive and diverse ecosystems, are currently threatened by a variety of stressors that result in increased prevalence of both bleaching and disease. Therefore, understanding the molecular mechanisms involved in these responses is critical to mitigate future damage to the reefs. One group of genes that is potentially involved in cnidarian immunity and symbiosis is GTPases of immunity associated proteins (GIMAP). In vertebrates, this family of proteins is involved in regulating the fate of developing lymphocytes and interacts with proteins involved in apoptosis and autophagy. As apoptosis, autophagy and immunity have previously been shown to be involved in cnidarian symbiosis and disease, the goal of this research was to determine the role of cnidarian GIMAPs in these processes using the anemone Exaiptasia pallida To do so, GIMAP genes were characterized in the E. pallida genome and changes in gene expression were measured using qPCR in response to chemical induction of apoptosis, autophagy and treatment with the immune stimulant lipopolysaccharide (LPS) in both aposymbiotic and symbiotic anemones. The results revealed four GIMAP-like genes in E. pallida, referred to as Ep_GIMAPs Induction of apoptosis and autophagy resulted in a general downregulation of Ep_GIMAPs, but no significant changes were observed in response to LPS treatment. This indicates that Ep_GIMAPs may be involved in the regulation of apoptosis and autophagy, and therefore could play a role in cnidarian-dinoflagellate symbiosis. Overall, these results increase our knowledge on the function of GIMAPs in a basal metazoan.

Transcription factor NF-κB in a basal metazoan, the sponge, has conserved and unique sequences, activities, and regulation

Herein, we characterize transcription factor NF-κB from the demosponge Amphimedon queenslandica (Aq). Aq–NF–κB is most similar to NF-κB p100/p105 among vertebrate proteins, with an N-terminal DNA-binding domain, a C-terminal Ankyrin (ANK) repeat domain, and a DNA binding-site profile akin to human NF-κB proteins. Like mammalian NF-κB p100, C-terminal truncation allows nuclear translocation of Aq–NF–κB and increases its transcriptional activation activity. Expression of IκB kinases (IKKs) induces proteasome-dependent C-terminal processing of Aq–NF–κB in human cells, and processing requires C-terminal serines in Aq–NF–κB. Unlike NF-κB p100, C-terminal sequences of Aq–NF–κB do not inhibit its DNA-binding activity. Tissue of a black encrusting demosponge contains NF-κB site DNA-binding activity, as well as nuclear and processed NF-κB. Treatment of sponge tissue with LPS increases both DNA-binding activity and processing of NF-κB. A. queenslandica transcriptomes contain homologs to upstream NF-κB pathway components. This is first functional characterization of NF-κB in sponge, the most basal multicellular animal.

Microbiota Differences of the Comb Jelly Mnemiopsis leidyi in Native and Invasive Sub-Populations

The translocation of non-indigenous species around the world, especially in marine systems, is a matter of concern for biodiversity conservation and ecosystem functioning. While specific traits are often recognized to influence the success in the establishment of non-indigenous species, the impact of the associated microbial community for the fitness, performance and invasion success of basal marine metazoans remains vastly unknown. In this study, we compared bacterial composition patterns of the invasive ctenophore Mnemiopsis leidyi in different native and invasive sub-populations along with the genetic structure of the host (polymorphic microsatellite markers). Amplicon sequencing of the 16S rRNA gene (V1-V2 hypervariable regions) revealed that M. leidyi as representative of the phylum Ctenophora, the sister group to all metazoans, harbored a distinct microbiota compared to the ambient seawater, which significantly differed across two major tissues, namely epidermis and gastrodermis. Moreover, we identified significant differences in bacterial community compositions between native and invasive sub-populations of M. leidyi indicating, that the microbiota community is likely influenced by the genotypic background of the host.

Discovery of a receptor guanylate cyclase expressed in the sperm flagella of stony corals

The receptor guanylate cyclases (rGCs) in animals serve as sensitive chemoreceptors to detect both chemical and environmental cues. In reproduction, rGCs were shown to be expressed on sperm and serve as receptors for egg-derived sperm-activating and sperm-attracting factors in some echinoderms and mammals. However, sperm-associated rGCs have only been identified in some deuterostomes thus far, and it remains unclear how widely rGCs are utilized in metazoan reproduction. To address this issue, this study investigated the existence and expression of rGCs, particularly asking if rGCs are involved in the reproduction of a basal metazoan, phylum Cnidaria, using the stony coral Euphyllia ancora. Six paralogous rGCs were identified from a transcriptome database of E. ancora, and one of the rGCs, GC-A, was shown to be specifically expressed in the testis. Immunohistochemical analyses demonstrated that E. ancora GC-A protein was expressed in the spermatocytes and spermatids and eventually congregated on the sperm flagella during spermatogenesis. These findings suggest that GC-A may be involved in the regulation of sperm activity and/or functions (e.g., fertilization) in corals. This study is the first to perform molecular characterization of rGCs in cnidarians and provides evidence for the possible involvement of rGCs in the reproduction of basal metazoans.

Comparative neuroanatomy of ctenophores: Neural and muscular systems in Euplokamis dunlapae and related species

Ctenophora is an early-branching basal metazoan lineage, which may have evolved neurons and muscles independently from other animals. However, despite the profound diversity among ctenophores, basal neuroanatomical data are limited to representatives of two genera. Here, we describe the organization of neuromuscular systems in eight ctenophore species focusing on Euplokamis dunlapae-the representative of the lineage sister to all other ctenophores. Cydippids (Hormiphora hormiphora and Dryodora glandiformis) and lobates (Bolinopsis infundibulum and Mnemiopsis leidyi) were used as reference platforms to cover both morphological and ecological diversity within the phylum. We show that even with substantial environmental differences, the basal organization of neural systems is conserved among ctenophores. In all species, we detected two distributed neuronal subsystems: the subepithelial polygonal network and the mesogleal elements. Nevertheless, each species developed specific innovations in neural, muscular, and receptor systems. Most notable Euplokamis-specific features are the following: (a) Comb nerves with giant axons. These nerves directly coordinate the rapid escape response bypassing the central integrative structure known as the aboral sensory organ. (b) Neural processes in tentacles along the rows of "boxes" providing structural support and located under striated muscles. (c) Radial muscles that cross the mesoglea and connect the outer wall to the aboral canal. (d) Flat muscles, encircling each meridional canal. Also, we detected a structurally different rectangular neural network in the feeding lobes of Lobata (Mnemiopsis/Bolinopsis) but not in other species. The described lineage-specific innovations can be used for future single-cell atlases of ctenophores and analyses of neuronal evolution in basal metazoans.

CTENO64 Is Required for Coordinated Paddling of Ciliary Comb Plate in Ctenophores.

Ctenophores, or comb jellies, are one of the earliest branching basal metazoan groups, whose phylogenetic position continues to be controversial. They have eight rows of iridescent structures, called comb plates, which are huge multiciliated paddle-like structures used for locomotion and uniquely found in this group of animals [1]. Despite a number of morphological and physiological studies over the past 50 years, the molecular nature of comb plates remains completely unknown. Here, we identified a protein CTENO64 that is specifically localized in the comb plates. This protein is only found in ctenophores and not in other animals or eukaryotic species that possess multiciliary cells or tissues. It is localized to regions, called compartmenting lamella (CL), which are uniquely seen in ctenophore multicilia, connecting adjacent cilia in the comb plates. Knockdown of the CTENO64 gene did not affect the formation of comb plates but caused the loss or misformation of CLs and the disruption of ciliary orientation, resulting in aberrant and non-planar waveforms in the mid-distal region of the comb plates. We propose that CLs have been convergently acquired in ctenophores to overcome the hydrodynamic constraints of possessing extremely long multicilia. Our findings provide the initial step in unveiling the molecular structure and evolutionary significance of ciliary comb plates and shed light not only on the hidden biology of ctenophores but also on the unique evolutionary pathway of these animals. VIDEO ABSTRACT.

Differentiation of the Oral–Aboral Axis and Body Parts during Life Cycle Transitions in Scyphozoa

Abstract—Differentiation of body axes during formation of primary body plan is one of the most fundamental processes in metazoan development. How did body axes emerge in the course of animal evolution? How did developmental processes associated with axes differentiation evolve? The basal metazoans, representatives of the phylum Cnidaria, are valuable objects for resolving these questions, since they have an evolutionarily simple body plan and only one (oral-aboral) body axis. The high abundance and diversity of the modes of asexual reproduction is an interesting feature of this group. In this review, we summarized the data on the establishment of primary body axis and differentiation of main morphological structures during asexual reproduction in representatives of the class Scyphozoa. We found that the relative orientation of the parental and offspring body axes varies significantly and depends on the mode of asexual reproduction, and that the same body parts of the offspring polyp or medusa can be formed from different parts of the parental organism. The high plasticity of scyphozoan developmental processes can be explained by assuming that various types of asexual reproduction evolved from regeneration processes in this group. To test this assumption, it is necessary to study the molecular mechanisms of differentiation of the primary body axis and the body parts during regeneration and asexual reproduction of scyphozoans.

Evolution of the Cholesterol Biosynthesis Pathway in Animals.

Cholesterol plays essential roles in animal development and disease progression. Here, we characterize the evolutionary pattern of the canonical cholesterol biosynthesis pathway (CBP) in the animal kingdom using both genome-wide analyses and functional experiments. CBP genes in the basal metazoans were inherited from their last common eukaryotic ancestor and evolutionarily conserved for cholesterol biosynthesis. The genomes of both the basal metazoans and deuterostomes retain almost the full set of CBP genes, while Cnidaria and many protostomes have independently experienced multiple massive losses of CBP genes that might be due to the geologic events during the Ediacaran period, such as the appearance of an exogenous sterol supply and the frequent perturbation of ocean oxygenation. Meanwhile, the indispensable utilization processes of cholesterol potentially strengthened the maintenance of the complete set of CBP genes in vertebrates. These results strengthen both biotic and abiotic roles in the macroevolution of a biosynthesis pathway in animals.

Mapping the Whole-Body Muscle Activity of Hydra vulgaris

Hydra is a cnidarian polyp with an anatomically simple neuromuscular system that can offer evolutionary insights on the functional design of animal body plans. Using calcium imaging to map the activity of the entire epitheliomuscular system of behaving Hydra, we find seven basic spatiotemporal patterns of muscle activity. Patterns include global and local activation events with widely varying kinetics of initiation and wave-like propagation. The orthogonally oriented endodermal and ectodermal muscle fibers are jointly activated during longitudinal contractions. Individual epitheliomuscular cells can participate in multiple patterns, even with very different kinetics. This cellular multifunctionality could enable the structurally simple epitheliomuscular tissue of basal metazoans to implement a diverse behavioral output.

Hypothesized Evolutionary Consequences of the Alternative Oxidase (AOX) in Animal Mitochondria.

The environment in which eukaryotes first evolved was drastically different from what they experience today, and one of the key limiting factors was the availability of oxygen for mitochondrial respiration. During the transition to a fully oxygenated Earth, other compounds such as sulfide posed a considerable constraint on using mitochondrial aerobic respiration for energy production. The ancestors of animals, and those that first evolved from the simpler eukaryotes have mitochondrial respiratory components that are absent from later-evolving animals. Specifically, mitochondria of most basal metazoans have a sulfide-resistant alternative oxidase (AOX), which provides a secondary oxidative pathway to the classical cytochrome pathway. In this essay, I argue that because of its resistance to sulfide, AOX respiration was critical to the evolution of animals by enabling oxidative metabolism under otherwise inhibitory conditions. I hypothesize that AOX allowed for metabolic flexibility during the stochastic oxygen environment of early Earth which shaped the evolution of basal metazoans. I briefly describe the known functions of AOX, with a particular focus on the decreased production of reactive oxygen species (ROS) during stress conditions. Then, I propose three evolutionary consequences of AOX-mediated protection from ROS observed in basal metazoans: 1) adaptation to stressful environments, 2) the persistence of facultative sexual reproduction, and 3) decreased mitochondrial DNA mutation rates. Recognizing the diversity of mitochondrial respiratory systems present in animals may help resolve the mechanisms involved in major evolutionary processes such as adaptation and speciation.

Разнообразие клеточных типов у гаплотипа H4 Placozoa sp.

Эволюционная ветвь Пластинчатых (Placozoa) имеет большое значение для понимания происхождения и эволюции Metazoa. Представители типа Placozoa обладают простым строением тела, однако полное отсутствие органов, нервной системы и мышечных клеток резко контрастирует с достаточно сложными поведенческими реакциями, в том числе динамическими изменениями формы тела. Известно 19 гаплотипов Placozoa, включающих два вида — Trichoplax adhaerens (H1) и Hoilungia hongkongensis (H13) — и пока ещё малоисследованные штаммы, обозначаемые как H2–H19 Placozoa sp. Хотя все Placozoa имеют похожий фенотип (пластинчатую дископодобную форму тела), анализ клеточной организации был сделан только для H1 (Trichoplax adhaerens) с идентификацией шести типов клеток. В этой работе мы, используя конфокальную и электронную микроскопию, исследовали организацию штамма H4, который близок к новому роду — Hoilungia. У H4 обнаружены все шесть основных типов клеток, идентифицированных ранее у H1. Между тем мы выявили у штамма H4 большее разнообразие клеток по форме, плотности и, впервые, по мембранному потенциалу митохондрий, чем ранее было описано для H1. Это позволяет нам подчеркнуть важность объединения методов молекулярно-генетических исследований и микроскопии для уточнения клеточной систематики, включая параметры митохондриальной активности для классификации клеток и их состояний. Сравнительно-эволюционное изучение клеточных типов у разных экологических групп Placozoa и их сопоставление с функциями расширяют понимание того, как относительно простой организм формирует сложное поведение и как типы клеток эволюционируют.

Neural system and receptor diversity in the ctenophore Beroe abyssicola.

Although, neurosensory systems might have evolved independently in ctenophores, very little is known about their organization and functions. Most ctenophores are pelagic and deep-water species and cannot be bred in the laboratory. Thus, it is not surprising that neuroanatomical data are available for only one genus within the group-Pleurobrachia. Here, using immunohistochemistry and scanning electron microscopy, we describe the organization of two distinct neural subsystems (subepithelial and mesogleal) and the structure of different receptor types in the comb jelly Beroe abyssicola-the voracious predator from North Pacific. A complex subepithelial neural network of Beroe, with five receptor types, covers the entire body surface and expands deep into the pharynx. Three types of mesogleal neurons are comparable to the cydippid Pleurobrachia. The predatory lifestyle of Beroe is supported by the extensive development of ciliated and muscular structures including the presence of giant muscles and feeding macrocilia. The obtained cell-type atlas illustrates different examples of lineage-specific innovations within these enigmatic marine animals and reveals the remarkable complexity of sensory and effector systems in this clade of basal Metazoa.

Establishment of the Axial Polarity and Cell Fate in Metazoa via Canonical Wnt Signaling: New Insights from Sponges and Annelids

In this paper, the role of the Wnt/β-catenin pathway in the establishment of the body plan in multicellular animals has been analyzed. We demonstrated that at different stages of development the Wnt signaling cascade is involved in several directly unrelated processes such as inner germ layer specification, anterior–posterior axis patterning, and terminal growth. Bioinformatics analysis revealed the independent divergence of Wnt ligands in basal Metazoa and conservation of Wnt subfamilies within the Cnidaria + Bilateria clade. Polarized expression of Wnt ligands in sponge and annelid larvae has been shown, thus confirming the applicability of the existing model of the main body axis patterning to two more taxa. The β-catenin localization and inhibitory analysis have provided evidence of the unlikely role for Wnt pathway in endomesoderm determination in nereid polychaetes. The evolutionary trend to establish alternative mechanisms even for key events such as segregation of germ layers has been observed in the case of more determinative ontogeny and predominance of autonomous specification, as exemplified by the heteroquadrant (unequal) type of spiral development.

Role of gene body methylation in acclimatization and adaptation in a basal metazoan

Gene body methylation (GBM) has been hypothesized to modulate responses to environmental change, including transgenerational plasticity, but the evidence thus far has been lacking. Here we show that coral fragments reciprocally transplanted between two distant reefs respond predominantly by increase or decrease in genome-wide GBM disparity: The range of methylation levels between lowly and highly methylated genes becomes either wider or narrower. Remarkably, at a broad functional level this simple adjustment correlated very well with gene expression change, reflecting a shifting balance between expressions of environmentally responsive and housekeeping genes. In our experiment, corals in a lower-quality habitat up-regulated genes involved in environmental responses, while corals in a higher-quality habitat invested more in housekeeping genes. Transplanted fragments showing closer GBM match to local corals attained higher fitness characteristics, which supports GBM's role in acclimatization. Fixed differences in GBM between populations did not align with plastic GBM changes and were mostly observed in genes with elevated FST, which suggests that they arose predominantly through genetic divergence. However, we cannot completely rule out transgenerational inheritance of acquired GBM states.