Conserved and divergent genes in apex and axis development of cnidarians

Despite their radial organization and their sister group position in the life tree, cnidarian species express during morphogenesis a large number of genes that are related to bilaterian developmental genes. Among those, homologs to forkhead, emx, aristaless, goosecoid, brachyury, wnt and nanos genes are regulated during apical patterning in cnidarians, suggesting that key components of early organizer activity were conserved across evolution and recruited for either anterior, axial, or dorso-ventral patterning in bilaterians. In contrast, the expression patterns of the cnidarian Hox-related genes suggest that the apical-basal axis of the cnidarian polyp and the anterior-posterior axis of bilaterians do not differentiate following homologous processes.     

Do cnidarians have a ParaHox cluster? Analysis of synteny around a Nematostella homeobox gene cluster

SUMMARY The Hox gene cluster is renowned for its role in developmental patterning of embryogenesis along the anterior–posterior axis of bilaterians. Its supposed evolutionary sister or paralog, the ParaHox cluster, is composed of Gsx, Xlox, and Cdx, and also has important roles in anterior–posterior development. There is a debate as to whether the cnidarians, as an outgroup to bilaterians, contain true Hox and ParaHox genes, or instead the Hox‐like gene complement of cnidarians arose from independent duplications to those that generated the genes of the bilaterian Hox and ParaHox clusters. A recent whole genome analysis of the cnidarian Nematostella vectensis found conserved synteny between this cnidarian and vertebrates, including a region of synteny between the putative Hox cluster of N. vectensis and the Hox clusters of vertebrates. No syntenic region was identified around a potential cnidarian ParaHox cluster. Here we use different approaches to identify a genomic region in N. vectensis that is syntenic with the bilaterian ParaHox cluster. This proves that the duplication that gave rise to the Hox and ParaHox regions of bilaterians occurred before the origin of cnidarians, and the cnidarian N. vectensis has bona fide Hox and ParaHox loci.     

Stem cell dynamics in Cnidaria: are there unifying principles?

The study of stem cells in cnidarians has a history spanning hundreds of years, but it has primarily focused on the hydrozoan genus Hydra. While Hydra has a number of self-renewing cell types that act much like stem cells—in particular the interstitial cell line—finding cellular homologues outside of the Hydrozoa has been complicated by the morphological simplicity of stem cells and inconclusive gene expression data. In non-hydrozoan cnidarians, an enigmatic cell type known as the amoebocyte might play a similar role to interstitial cells, but there is little evidence that I-cells and amoebocytes are homologous. Instead, self-renewal and transdifferentiation of epithelial cells was probably more important to ancestral cnidarian development than any undifferentiated cell lineage, and only later in evolution did one or more cell types come under the regulation of a “stem” cell line. Ultimately, this hypothesis and competing ones will need to be tested by expanding genetic and developmental studies on a variety of cnidarian model systems.     

Nervous systems of the sea anemone Nematostella vectensis are generated by ectoderm and endoderm and shaped by distinct mechanisms

As a sister group to Bilateria, Cnidaria is important for understanding early nervous system evolution. Here we examine neural development in the anthozoan cnidarian Nematostella vectensis in order to better understand whether similar developmental mechanisms are utilized to establish the strikingly different overall organization of bilaterian and cnidarian nervous systems. We generated a neuron-specific transgenic NvElav1 reporter line of N. vectensis and used it in combination with immunohistochemistry against neuropeptides, in situ hybridization and confocal microscopy to analyze nervous system formation in this cnidarian model organism in detail. We show that the development of neurons commences in the ectoderm during gastrulation and involves interkinetic nuclear migration. Transplantation experiments reveal that sensory and ganglion cells are autonomously generated by the ectoderm. In contrast to bilaterians, neurons are also generated throughout the endoderm during planula stages. Morpholino-mediated gene knockdown shows that the development of a subset of ectodermal neurons requires NvElav1, the ortholog to bilaterian neural elav1 genes. The orientation of ectodermal neurites changes during planula development from longitudinal (in early-born neurons) to transverse (in late-born neurons), whereas endodermal neurites can grow in both orientations at any stage. Our findings imply that elav1-dependent ectodermal neurogenesis evolved prior to the divergence of Cnidaria and Bilateria. Moreover, they suggest that, in contrast to bilaterians, almost the entire ectoderm and endoderm of the body column of Nematostella planulae have neurogenic potential and that the establishment of connectivity in its seemingly simple nervous system involves multiple neurite guidance systems.     

Expanded Functional Diversity of Shaker K+ Channels in Cnidarians Is Driven by Gene Expansion

The genome of the cnidarian Nematostella vectensis (starlet sea anemone) provides a molecular genetic view into the first nervous systems, which appeared in a late common ancestor of cnidarians and bilaterians. Nematostella has a surprisingly large and diverse set of neuronal signaling genes including paralogs of most neuronal signaling molecules found in higher metazoans. Several ion channel gene families are highly expanded in the sea anemone, including three subfamilies of the Shaker K⁺ channel gene family: Shaker (Kv1), Shaw (Kv3) and Shal (Kv4). In order to better understand the physiological significance of these voltage-gated K⁺ channel expansions, we analyzed the function of 18 members of the 20 gene Shaker subfamily in Nematostella. Six of the Nematostella Shaker genes express functional homotetrameric K⁺ channels in vitro. These include functional orthologs of bilaterian Shakers and channels with an unusually high threshold for voltage activation. We identified 11 Nematostella Shaker genes with a distinct “silent” or “regulatory” phenotype; these encode subunits that function only in heteromeric channels and serve to further diversify Nematostella Shaker channel gating properties. Subunits with the regulatory phenotype have not previously been found in the Shaker subfamily, but have evolved independently in the Shab (Kv2) family in vertebrates and the Shal family in a cnidarian. Phylogenetic analysis indicates that regulatory subunits were present in ancestral cnidarians, but have continued to diversity at a high rate after the split between anthozoans and hydrozoans. Comparison of Shaker family gene complements from diverse metazoan species reveals frequent, large scale duplication has produced highly unique sets of Shaker channels in the major metazoan lineages.     

Regionalized nervous system in Hydra and the mechanism of its development

The last common ancestor of Bilateria and Cnidaria is considered to develop a nervous system over 500 million years ago. Despite the long course of evolution, many of the neuron-related genes, which are active in Bilateria, are also found in the cnidarian Hydra. Thus, Hydra is a good model to study the putative primitive nervous system in the last common ancestor that had the great potential to evolve to a more advanced one. Regionalization of the nervous system is one of the advanced features of bilaterian nervous system. Although a regionalized nervous system is already known to be present in Hydra, its developmental mechanisms are poorly understood. In this study we show how it is formed and maintained, focusing on the neuropeptide Hym-176 gene and its paralogs. First, we demonstrate that four axially localized neuron subsets that express different combination of the neuropeptide Hym-176 gene and its paralogs cover almost an entire body, forming a regionalized nervous system in Hydra. Second, we show that positional information governed by the Wnt signaling pathway plays a key role in determining the regional specificity of the neuron subsets as is the case in bilaterians. Finally, we demonstrated two basic mechanisms, regionally restricted new differentiation and phenotypic conversion, both of which are in part conserved in bilaterians, are involved in maintaining boundaries between the neuron subsets. Therefore, this study is the first comprehensive analysis of the anatomy and developmental regulation of the divergently evolved and axially regionalized peptidergic nervous system in Hydra, implicating an ancestral origin of neural regionalization.     

Independent origins of neurons and synapses: insights from ctenophores

There is more than one way to develop neuronal complexity, and animals frequently use different molecular toolkits to achieve similar functional outcomes. Genomics and metabolomics data from basal metazoans suggest that neural signalling evolved independently in ctenophores and cnidarians/bilaterians. This polygenesis hypothesis explains the lack of pan-neuronal and pan-synaptic genes across metazoans, including remarkable examples of lineage-specific evolution of neurogenic and signalling molecules as well as synaptic components. Sponges and placozoans are two lineages without neural and muscular systems. The possibility of secondary loss of neurons and synapses in the Porifera/Placozoa clades is a highly unlikely and less parsimonious scenario. We conclude that acetylcholine, serotonin, histamine, dopamine, octopamine and gamma-aminobutyric acid (GABA) were recruited as transmitters in the neural systems in cnidarian and bilaterian lineages. By contrast, ctenophores independently evolved numerous secretory peptides, indicating extensive adaptations within the clade and suggesting that early neural systems might be peptidergic. Comparative analysis of glutamate signalling also shows numerous lineage-specific innovations, implying the extensive use of this ubiquitous metabolite and intercellular messenger over the course of convergent and parallel evolution of mechanisms of intercellular communication. Therefore: (i) we view a neuron as a functional character but not a genetic character, and (ii) any given neural system cannot be considered as a single character because it is composed of different cell lineages with distinct genealogies, origins and evolutionary histories. Thus, when reconstructing the evolution of nervous systems, we ought to start with the identification of particular cell lineages by establishing distant neural homologies or examples of convergent evolution. In a corollary of the hypothesis of the independent origins of neurons, our analyses suggest that both electrical and chemical synapses evolved more than once.     

A fragile X mental retardation-like gene in a cnidarian

The fragile X mental retardation syndrome in humans is caused by a mutational loss of function of the fragile X mental retardation gene 1 (FMR1). FMR1 is an RNA-binding protein, involved in the development and function of the nervous system. Despite of its medical significance, the evolutionary origin of FMR1 has been unclear. Here, we report the molecular characterization of HyFMR1, an FMR1 orthologue, from the cnidarian hydroid Hydractinia echinata. Cnidarians are the most basal metazoans possessing neurons. HyFMR1 is expressed throughout the life cycle of Hydractinia. Its expression pattern correlates to the position of neurons and their precursor stem cells in the animal. Our data indicate that the origin of the fraxile X related (FXR) protein family dates back at least to the common ancestor of cnidarians and bilaterians. The lack of FXR proteins in other invertebrates may have been due to gene loss in particular lineages.     

Evidence for Involvement of Wnt Signalling in Body Polarities,Cell Proliferation,and the Neuro-Sensory System in an Adult Ctenophore

Signalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/β-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular β-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures.     

A non-tree-based comprehensive study of metazoan Hox and ParaHox genes prompts new insights into their origin and evolution

Background  

Hox and the closely-related ParaHox genes, which emerged prior to the divergence between cnidarians and bilaterians, are the most well-known members of the ancient genetic toolkit that controls embryonic development across all metazoans. Fundamental questions relative to their origin and evolutionary relationships remain however unresolved. We investigate here the evolution of metazoan Hox and ParaHox genes using the HoxPred program that allows the identification of Hox genes without the need of phylogenetic tree reconstructions.     

Evolutionary origin of rhopalia: insights from cellular‐level analyses of Otx and POU expression patterns in the developing rhopalial nervous system

SUMMARY In Cnidaria, the medusae of Scyphozoa and its sister‐group Cubozoa uniquely possess rhopalia at their bell margin. These sensory centers coordinate behavior and development. We used fluorescent in situ hybridization and confocal microscopy to examine mRNA expression patterns in Aurelia sp.1 (Cnidaria, Scyphozoa) during early medusa formation, while simultaneously visualizing the developing nervous system by immunofluorescence. The genes investigated include AurOtx1, and the POU genes, AurPit1, and AurBrn3, homologs of genes known to function in cephalar neural organization and sensory cell differentiation across Bilateria. Our results show that AurOtx1 expression defines the major part of the oral neuroectodermal domain of the rhopalium, within which distinct populations of AurBrn3‐ and AurPit1‐expressing sensory cells develop. Thus, despite the unique attributes of rhopalial evolution, we suggest that the rhopalial nervous system of scyphozoan medusae involves similar patterns of differential expression of genes that function in bilaterian cephalic structure and neuroendocrine system development. We propose that rhopalia evolved from preexisting sensory structures that developed distinct populations of sensory cells differentially expressing POU genes within Otx oral‐neuroectodermal domains. This implies some commonality of developmental genetic functions involving these genes in the still poorly constrained common ancestor of bilaterians and cnidarians.     

The evolutionary history of vertebrate cranial placodes – I: Cell type evolution

Vertebrate cranial placodes are crucial contributors to the vertebrate cranial sensory apparatus. Their evolutionary origin has attracted much attention from evolutionary and developmental biologists, yielding speculation and hypotheses concerning their putative homologues in other lineages and the developmental and genetic innovations that might have underlain their origin and diversification. In this article we first briefly review our current understanding of placode development and the cell types and structures they form. We next summarise previous hypotheses of placode evolution, discussing their strengths and caveats, before considering the evolutionary history of the various cell types that develop from placodes. In an accompanying review, we also further consider the evolution of ectodermal patterning. Drawing on data from vertebrates, tunicates, amphioxus, other bilaterians and cnidarians, we build these strands into a scenario of placode evolutionary history and of the genes, cells and developmental processes that underlie placode evolution and development.     

Myxosporea (Myxozoa,Cnidaria) Lack DNA Cytosine Methylation

DNA cytosine methylation is central to many biological processes, including regulation of gene expression, cellular differentiation, and development. This DNA modification is conserved across animals, having been found in representatives of sponges, ctenophores, cnidarians, and bilaterians, and with very few known instances of secondary loss in animals. Myxozoans are a group of microscopic, obligate endoparasitic cnidarians that have lost many genes over the course of their evolution from free-living ancestors. Here, we investigated the evolution of the key enzymes involved in DNA cytosine methylation in 29 cnidarians and found that these enzymes were lost in an ancestor of Myxosporea (the most speciose class of Myxozoa). Additionally, using whole-genome bisulfite sequencing, we confirmed that the genomes of two distant species of myxosporeans, Ceratonova shasta and Henneguya salminicola, completely lack DNA cytosine methylation. Our results add a notable and novel taxonomic group, the Myxosporea, to the very short list of animal taxa lacking DNA cytosine methylation, further illuminating the complex evolutionary history of this epigenetic regulatory mechanism.     

Missing link in the evolution of Hox clusters

Hox cluster has key roles in regulating the patterning of the antero-posterior axis in a metazoan embryo. It consists of the anterior, central and posterior genes; the central genes have been identified only in bilaterians, but not in cnidarians, and are responsible for archiving morphological complexity in bilaterian development. However, their evolutionary history has not been revealed, that is, there has been a "missing link". Here we show the evolutionary history of Hox clusters of 18 bilaterians and 2 cnidarians by using a new method, "motif-based reconstruction", examining the gain/loss processes of evolutionarily conserved sequences, "motifs", outside the homeodomain. We successfully identified the missing link in the evolution of Hox clusters between the cnidarian-bilaterian ancestor and the bilaterians as the ancestor of the central genes, which we call the proto-central gene. Exploring the correspondent gene with the proto-central gene, we found that one of the acoela Hox genes has the same motif repertory as that of the proto-central gene. This interesting finding suggests that the acoela Hox cluster corresponds with the missing link in the evolution of the Hox cluster between the cnidarian-bilaterian ancestor and the bilaterians. Our findings suggested that motif gains/diversifications led to the explosive diversity of the bilaterian body plan.     

Evolutionary crossroads in developmental biology: Cnidaria

There is growing interest in the use of cnidarians (corals, sea anemones, jellyfish and hydroids) to investigate the evolution of key aspects of animal development, such as the formation of the third germ layer (mesoderm), the nervous system and the generation of bilaterality. The recent sequencing of the Nematostella and Hydra genomes, and the establishment of methods for manipulating gene expression, have inspired new research efforts using cnidarians. Here, we present the main features of cnidarian models and their advantages for research, and summarize key recent findings using these models that have informed our understanding of the evolution of the developmental processes underlying metazoan body plan formation.     

A novel minicollagen gene links cnidarians and myxozoans

Myxozoans are enigmatic endoparasitic organisms sharing morphological features with bilateria, protists and cnidarians. This, coupled with their highly divergent gene sequences, has greatly obscured their phylogenetic affinities. Here we report the sequencing and characterization of a minicollagen homologue (designated Tb-Ncol-1) in the myxozoan Tetracapsuloides bryosalmonae. Minicollagens are phylum-specific genes encoding cnidarian nematocyst proteins. Sequence analysis revealed a cysteine-rich domain (CRD) architecture and genomic organization similar to group 1 minicollagens. Homology modelling predicted similar three-dimensional structures to Hydra CRDs despite deviations from the canonical pattern of group 1 minicollagens. The discovery of this minicollagen gene strongly supports myxozoans as cnidarians that have radiated as endoparasites of freshwater, marine and terrestrial hosts. It also reveals novel protein sequence variation of relevance to understanding the evolution of nematocyst complexity, and indicates a molecular/morphological link between myxozoan polar capsules and cnidarian nematocysts. Our study is the first to illustrate the power of using genes related to a taxon-specific novelty for phylogenetic inference within the Metazoa, and it exemplifies how the evolutionary relationships of other metazoans characterized by extreme sequence divergence could be similarly resolved.