Expression Analysis of Cnidarian-Specific Neuropeptides in a Sea Anemone Unveils an Apical-Organ-Associated Nerve Net That Disintegrates at Metamorphosis.
ABSTRACT: Neuropeptides are ancient neuronal signaling molecules that have diversified across Cnidaria (e.g., jellyfish, corals, and sea anemones) and its sister group Bilateria (e.g., vertebrates, insects, and worms). Over the course of neuropeptide evolution emerged lineage-specific neuropeptides, but their roles in the evolution and diversification of nervous system function remain enigmatic. As a step toward filling in this knowledge gap, we investigated the expression pattern of a cnidarian-specific neuropeptide-RPamide-during the development of the starlet sea anemone Nematostella vectensis, using in situ hybridization and immunohistochemistry. We show that RPamide precursor transcripts first occur during gastrulation in scattered epithelial cells of the aboral ectoderm. These RPamide-positive epithelial cells exhibit a spindle-shaped, sensory-cell-like morphology, and extend basal neuronal processes that form a nerve net in the aboral ectoderm of the free-swimming planula larva. At the aboral end, RPamide-positive sensory cells become integrated into the developing apical organ that forms a bundle of long cilia referred to as the apical tuft. Later during planula development, RPamide expression becomes evident in sensory cells in the oral ectoderm of the body column and pharynx, and in the developing endodermal nervous system. At metamorphosis into a polyp, the RPamide-positive sensory nerve net in the aboral ectoderm degenerates by apoptosis, and RPamide expression begins in ectodermal sensory cells of growing oral tentacles. In addition, we find that the expression pattern of RPamide in planulae differs from that of conserved neuropeptides that are shared across Cnidaria and Bilateria, indicative of distinct functions. Our results not only provide the anatomical framework necessary to analyze the function of the cnidarian-specific neuropeptides in future studies, but also reveal previously unrecognized features of the sea anemone nervous system-the apical organ neurons of the planula larva, and metamorphosis-associated reorganization of the ectodermal nervous system.
Project description:Neuropeptides are evolutionarily ancient peptide hormones of the nervous and neuroendocrine systems, and are thought to have regulated metamorphosis in early animal ancestors. In particular, the deeply conserved Wamide family of neuropeptides-shared across Bilateria (e.g. insects and worms) and its sister group Cnidaria (e.g. jellyfishes and corals)-has been implicated in mediating life-cycle transitions, yet their endogenous roles remain poorly understood. By using CRISPR-Cas9-mediated reverse genetics, we show that cnidarian Wamide-referred to as GLWamide-regulates the timing of life cycle transition in the sea anemone cnidarian Nematostella vectensis. We find that mutant planula larvae lacking GLWamides transform into morphologically normal polyps at a rate slower than that of the wildtype control larvae. Treatment of GLWamide null mutant larvae with synthetic GLWamide peptides is sufficient to restore a normal rate of metamorphosis. These results demonstrate that GLWamide plays a dispensable, modulatory role in accelerating metamorphosis in a sea anemone.
Project description:BACKGROUND:zic genes are members of the gli/glis/nkl/zic super-family of C2H2 zinc finger (ZF) transcription factors. Homologs of the zic family have been implicated in patterning neural and mesodermal tissues in bilaterians. Prior to this study, the origin of the metazoan zic gene family was unknown and expression of zic gene homologs during the development of early branching metazoans had not been investigated. RESULTS:Phylogenetic analyses of novel zic candidate genes identified a definitive zic homolog in the placozoan Trichoplax adhaerens, two gli/glis/nkl-like genes in the ctenophore Mnemiopsis leidyi, confirmed the presence of three gli/glis/nkl-like genes in Porifera, and confirmed the five previously identified zic genes in the cnidarian Nematostella vectensis. In the cnidarian N. vectensis, zic homologs are expressed in ectoderm and the gastrodermis (a bifunctional endomesoderm), in presumptive and developing tentacles, and in oral and sensory apical tuft ectoderm. The Capitella teleta zic homolog (Ct-zic) is detectable in a subset of the developing nervous system, the foregut, and the mesoderm associated with the segmentally repeated chaetae. Lastly, expression of gli and glis homologs in Mnemiopsis. leidyi is detected exclusively in neural cells in floor of the apical organ. CONCLUSIONS:Based on our analyses, we propose that the zic gene family arose in the common ancestor of the Placozoa, Cnidaria and Bilateria from a gli/glis/nkl-like gene and that both ZOC and ZF-NC domains evolved prior to cnidarian-bilaterian divergence. We also conclude that zic expression in neural ectoderm and developing neurons is pervasive throughout the Metazoa and likely evolved from neural expression of an ancestral gli/glis/nkl/zic gene. zic expression in bilaterian mesoderm may be related to the expression in the gastrodermis of a cnidarian-bilaterian common ancestor.
Project description:The spatial transcriptome data has allowed us to explore the differential gene expression among the oral-aboral domains and provided new insights into the apical organ signalling pathways for future mechanistic studies and genes associated with oral/aboral differentiation. This resource identifies numerous candidates for elucidating the functional role of the apical organ and to address the evolution of the metazoan larval nervous system. Overall design: Here, we uncovered the molecular signature of the apical organ in N. vectensis through spatial transcriptomics. We performed microdissection on N. vectensis larvae and carefully separated the aboral tissue from the rest of the larval body at the planula and late planula development stages.We acquired transcriptome data from both the aboral tissue and the rest of the body separately to perform differential gene expression (DGE) analysis.
Project description:The fibroblast growth factor (FGF) signal transduction pathway serves as one of the key regulators of early metazoan development, displaying conserved roles in the specification of endodermal, mesodermal, and neural fates during vertebrate development. FGF signals also regulate gastrulation, in part, by triggering epithelial to mesenchymal transitions in embryos of both vertebrates and invertebrates. Thus, FGF signals coordinate gastrulation movements across many different phyla. To help understand the breadth of FGF signaling deployment across the animal kingdom, we have examined the presence and expression of genes encoding FGF pathway components in the anthozoan cnidarian Nematostella vectensis. We isolated three FGF ligands (NvFGF8A, NvFGF8B, and NvFGF1A), two FGF receptors (NvFGFRa and NvFGFRb), and two orthologs of vertebrate FGF responsive genes, Sprouty (NvSprouty), an inhibitor of FGF signaling, and Churchill (NvChurchill), a Zn finger transcription factor. We found these FGF ligands, receptors, and response gene expressed asymmetrically along the oral/aboral axis during gastrulation and in a developing chemosensory structure of planula stages known as the apical tuft. These results suggest a conserved role for FGF signaling molecules in coordinating both gastrulation and neural induction that predates the Cambrian explosion and the origins of the Bilateria.
Project description:Insect gustatory and odorant receptors (GRs and ORs) form a superfamily of novel transmembrane proteins, which are expressed in chemosensory neurons that detect environmental stimuli. Here we identify homologues of GRs (Gustatory receptor-like (Grl) genes) in genomes across Protostomia, Deuterostomia and non-Bilateria. Surprisingly, two Grls in the cnidarian Nematostella vectensis, NvecGrl1 and NvecGrl2, are expressed early in development, in the blastula and gastrula, but not at later stages when a putative chemosensory organ forms. NvecGrl1 transcripts are detected around the aboral pole, considered the equivalent to the head-forming region of Bilateria. Morpholino-mediated knockdown of NvecGrl1 causes developmental patterning defects of this region, leading to animals lacking the apical sensory organ. A deuterostome Grl from the sea urchin Strongylocentrotus purpuratus displays similar patterns of developmental expression. These results reveal an early evolutionary origin of the insect chemosensory receptor family and raise the possibility that their ancestral role was in embryonic development.
Project description:During animal evolution, ancestral Cnidaria and Bilateria diverged more than 600 million years ago. The nervous systems of extant cnidarians are strongly peptidergic. Neuropeptides have been isolated and sequenced from a few model cnidarians, but a global investigation of the presence of neuropeptides in all cnidarian classes has been lacking. Here, we have used a recently developed software program to annotate neuropeptides in the publicly available genomes and transcriptomes from members of the classes Cubozoa, Scyphozoa, and Staurozoa (which all belong to the subphylum Medusozoa) and contrasted these results with neuropeptides present in the subclass Octocorallia (belonging to the class Anthozoa). We found three to six neuropeptide preprohormone genes in members of the above-mentioned cnidarian classes or subclasses, each coding for several (up to thirty-two) similar or identical neuropeptide copies. Two of these neuropeptide preprohormone genes are present in all cnidarian classes/subclasses investigated, so they are good candidates for being among the first neuropeptide genes evolved in cnidarians. One of these primordial neuropeptide genes codes for neuropeptides having the C-terminal sequence GRFamide (pQGRFamide in Octocorallia; pQWLRGRFamide in Cubozoa and Scyphozoa; pQFLRGRFamide in Staurozoa). The other primordial neuropeptide gene codes for peptides having RPRSamide or closely resembling amino acid sequences. In addition to these two primordial neuropeptide sequences, cnidarians have their own class- or subclass-specific neuropeptides, which probably evolved to serve class/subclass-specific needs. When we carried out phylogenetic tree analyses of the GRFamide or RPRSamide preprohormones from cubozoans, scyphozoans, staurozoans, and octocorallia, we found that their phylogenetic relationships perfectly agreed with current models of the phylogeny of the studied cnidarian classes and subclasses. These results support the early origins of the GRFamide and RPRSamide preprohormone genes.
Project description:Formation of all metazoan bodies is controlled by a group of selector genes including homeobox genes, highly conserved across the entire animal kingdom. The homeobox genes from Pou and Six classes are key members of the regulation cascades determining development of sensory organs, nervous system, gonads and muscles. Besides using common bilaterian models, more attention has recently been targeted at the identification and characterization of these genes within the basal metazoan phyla. Cnidaria as a diploblastic sister group to bilateria with simple and yet specialized organs are suitable models for studies on the sensory organ origin and the associated role of homeobox genes. In this work, Pou and Six homeobox genes, together with a broad range of other sensory-specific transcription factors, were identified in the transcriptome of hydrozoan jellyfish Craspedacusta sowerbyi. Phylogenetic analyses of Pou and Six proteins revealed cnidarian-specific sequence motifs and contributed to the classification of individual factors. The majority of the Craspedacusta sowerbyi Pou and Six homeobox genes are predominantly expressed in statocysts, manubrium and nerve ring, the tissues with sensory and nervous activities. The described diversity and expression patterns of Pou and Six factors in hydrozoan jellyfish highlight their evolutionarily conserved functions. This study extends the knowledge of the cnidarian genome complexity and shows that the transcriptome of hydrozoan jellyfish is generally rich in homeodomain transcription factors employed in the regulation of sensory and nervous functions.
Project description:Abstract Neuropeptides are neurosecretory signaling molecules in protostomes and deuterostomes (together Nephrozoa). Little, however, is known about the neuropeptide complement of the sister group of Nephrozoa, the Xenacoelomorpha, which together form the Bilateria. Because members of the xenacoelomorph clades Xenoturbella, Nemertodermatida, and Acoela differ extensively in their central nervous system anatomy, the reconstruction of the xenacoelomorph and bilaterian neuropeptide complements may provide insights into the relationship between nervous system evolution and peptidergic signaling. Here, we analyzed transcriptomes of seven acoels, four nemertodermatids, and two Xenoturbella species using motif searches, similarity searches, mass spectrometry and phylogenetic analyses to characterize neuropeptide precursors and neuropeptide receptors. Our comparison of these repertoires with previously reported nephrozoan and cnidarian sequences shows that the majority of annotated neuropeptide GPCRs in cnidarians are not orthologs of specific bilaterian neuropeptide receptors, which suggests that most of the bilaterian neuropeptide systems evolved after the cnidarian–bilaterian evolutionary split. This expansion of more than 20 peptidergic systems in the stem leading to the Bilateria predates the evolution of complex nephrozoan organs and nervous system architectures. From this ancient set of neuropeptides, acoels show frequent losses that correlate with their divergent central nervous system anatomy. We furthermore detected the emergence of novel neuropeptides in xenacoelomorphs and their expansion along the nemertodermatid and acoel lineages, the two clades that evolved nervous system condensations. Together, our study provides fundamental insights into the early evolution of the bilaterian peptidergic systems, which will guide future functional and comparative studies of bilaterian nervous systems.
Project description:Conventional wisdom suggests that bilateral organisms arose from ancestors that were radially, rather than bilaterally, symmetrical and, therefore, had a single body axis and no mesoderm. The two main hypotheses on how this transformation took place consider either a simple organism akin to the planula larva of extant cnidarians or the acoel Platyhelminthes (planuloid-acoeloid theory), or a rather complex organism bearing several or most features of advanced coelomate bilaterians (archicoelomate theory). We report phylogenetic analyses of bilaterian metazoans using quantitative (ribosomal, nuclear and expressed sequence tag sequences) and qualitative (HOX cluster genes and microRNA sets) markers. The phylogenetic trees obtained corroborate the position of acoel and nemertodermatid flatworms as the earliest branching extant members of the Bilateria. Moreover, some acoelomate and pseudocoelomate clades appear as early branching lophotrochozoans and deuterostomes. These results strengthen the view that stem bilaterians were small, acoelomate/pseudocoelomate, benthic organisms derived from planuloid-like organisms. Because morphological and recent gene expression data suggest that cnidarians are actually bilateral, the origin of the last common bilaterian ancestor has to be put back in time earlier than the cnidarian-bilaterian split in the form of a planuloid animal. A new systematic scheme for the Bilateria that includes the Cnidaria is suggested and its main implications discussed.
Project description:The primary axis of cnidarians runs from the oral pole to the apical tuft and defines the major body axis of both the planula larva and adult polyp. In the anthozoan cnidarian Nematostella vectensis, the primary oral-aboral (O-Ab) axis first develops during the early embryonic stage. Here, we present evidence that pharmaceutical activators of canonical wnt signaling affect molecular patterning along the primary axis of Nematostella. Although not overtly morphologically complex, molecular investigations in Nematostella reveal that the O-Ab axis is demarcated by the expression of differentially localized signaling molecules and transcription factors that may serve roles in establishing distinct ectodermal domains. We have further characterized the larval epithelium by determining the position of a nested set of molecular boundaries, utilizing several newly characterized as well as previously reported epithelial markers along the primary axis. We have assayed shifts in their position in control embryos and in embryos treated with the pharmacological agents alsterpaullone and azakenpaullone, Gsk3? inhibitors that act as canonical wnt agonists, and the Wnt antagonist iCRT14, following gastrulation. Agonist drug treatments result in an absence of aboral markers, a shift in the expression boundaries of oral markers toward the aboral pole, and changes in the position of differentially localized populations of neurons in a dose-dependent manner, while antagonist treatment had the opposite effect. These experiments are consistent with canonical wnt signaling playing a role in an orally localized wnt signaling center. These findings suggest that in Nematostella, wnt signaling mediates O-Ab ectodermal patterning across a surprisingly complex epithelium in planula stages following gastrulation in addition to previously described roles for the wnt signaling pathway in endomesoderm specification during gastrulation and overall animal-vegetal patterning at earlier stages of anthozoan development.