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Background Light is essential for various biological activities. In particular, visual information through eyes or eyespots is very important for most of animals, and thus, the functions and developmental mechanisms of visual systems have been well studied to date. In addition, light-dependent non-visual systems expressing photoreceptor Opsins have been used to study the effects of light on diverse animal behaviors. However, it remains unclear how light-dependent systems were acquired and diversified during deuterostome evolution due to an almost complete lack of knowledge on the light-response signaling pathway in Ambulacraria, one of the major groups of deuterostomes and a sister group of chordates. Results Here, we show that sea urchin larvae utilize light for digestive tract activity. We found that photoirradiation of larvae induces pyloric opening even without addition of food stimuli. Micro-surgical and knockdown experiments revealed that this stimulating light is received and mediated by Go(/RGR)-Opsin (Opsin3.2 in sea urchin genomes) cells around the anterior neuroectoderm. Furthermore, we found that the anterior neuroectodermal serotoninergic neurons near Go-Opsin-expressing cells are essential for mediating light stimuli-induced nitric oxide (NO) release at the pylorus. Our results demonstrate that the light>Go-Opsin>serotonin>NO pathway functions in pyloric opening during larval stages. Conclusions The results shown here will lead us to understand how light-dependent systems of pyloric opening functioning via neurotransmitters were acquired and established during animal evolution. Based on the similarity of nervous system patterns and the gut proportions among Ambulacraria, we suggest the light>pyloric opening pathway may be conserved in the clade, although the light signaling pathway has so far not been reported in other members of the group. In light of brain-gut interactions previously found in vertebrates, we speculate that one primitive function of anterior neuroectodermal neurons (brain neurons) may have been to regulate the function of the digestive tract in the common ancestor of deuterostomes. Given that food consumption and nutrient absorption are essential for animals, the acquirement and development of brain-based sophisticated gut regulatory system might have been important for deuterostome evolution.

The ribonuclease A family of proteins is well studied from the biochemical and biophysical points of view, but its evolutionary origins are obscure, as no sequences homologous to this family have been reported outside of vertebrates. Recently, the spatial structure of the ribonuclease domain from a bacterial polymorphic toxin was shown to be closely similar to the structure of vertebrate ribonuclease A. The absence of sequence similarity between the two structures prompted a speculation of convergent evolution of bacterial and vertebrate ribonuclease A-like enzymes. We show that bacterial and homologous archaeal polymorphic toxin ribonucleases with a known or predicted ribonuclease A-like fold are distant homologs of the ribonucleases from the EndoU family, found in all domains of cellular life and in viruses. We also detected a homolog of vertebrate ribonucleases A in the transcriptome assembly of the sea urchin Mesocentrotus franciscanus. These observations argue for the common ancestry of prokaryotic ribonuclease A-like and ubiquitous EndoU-like ribonucleases, and suggest a better-grounded scenario for the origin of animal ribonucleases A, which could have emerged in the deuterostome lineage, either by an extensive modification of a copy of an EndoU gene, or, more likely, by a horizontal acquisition of a prokaryotic immunity-mediating ribonuclease gene.

Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single-cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identify 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these reveal a highly detailed portrait of cell diversity across the larva, including the identification of neuronal cell types. We then validate important gene regulatory networks driving sea urchin development and reveal new domains of activity within the larval body. Focusing on neurons that co-express Pdx-1 and Brn1/2/4, we identify an unprecedented number of genes shared by this population of neurons in sea urchin and vertebrate endocrine pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we show that Pdx1 is necessary for the acquisition of the neuronal identity of these cells. We hypothesize that a network similar to the one orchestrated by Pdx1 in the sea urchin neurons was active in an ancestral cell type and then inherited by neuronal and pancreatic developmental lineages in sea urchins and vertebrates.

DNA methylation (5-methylcytosine; 5mC) is a repressive gene-regulatory mark required for vertebrate embryogenesis. Genomic 5mC is tightly regulated through the coordinated action of DNA methyltransferases, which deposit 5mC, and TET enzymes, which participate in its active removal through the formation of 5-hydroxymethylcytosine (5hmC). TET enzymes are essential for mammalian gastrulation and activation of vertebrate developmental enhancers, however, to date, a clear picture of 5hmC function, abundance, and genomic distribution in non-vertebrate lineages is lacking. By employing base-resolution 5mC and 5hmC quantification during sea urchin and lancelet embryogenesis, we shed light on the roles of non-vertebrate 5hmC and TET enzymes. We find that these invertebrate deuterostomes employ TET enzymes for targeted demethylation of regulatory regions associated with developmental genes and show that the complement of identified 5hmC-regulated genes is conserved to vertebrates. This work thus demonstrates that active 5mC removal from regulatory regions is a common feature of deuterostome embryogenesis suggestive of unexpected deep conservation of a major gene-regulatory module.

Glycine is an important neurotransmitter in vertebrates, performing both excitatory and inhibitory actions. Synaptic levels of glycine are tightly controlled by the action of two glycine transporters, GlyT1 and GlyT2, located on the surface of glial cells and glycinergic or glutamatergic neurons, respectively. Glycinergic neurotransmission in invertebrates has so far only been investigated in a very limited number of species, and, although it was suggested that its functions are to some extent conserved with vertebrates, the evolution of glycinergic neurotransmission remains very poorly understood. Here, by combining phylogenetic and gene expression analyses, we characterized the glycine transporter complement of amphioxus, an important invertebrate model for studying the evolution of chordates. We show that amphioxus possesses three glycine transporter genes, two of which (GlyT2.1 and GlyT2.2) are closely related to GlyT2 of vertebrates, while the other (GlyT) is a member of an ancestral clade of deuterostome glycine transporters. While expression of GlyT2.2 is predominantly non-neural, GlyT and GlyT2.1 are widely expressed in the amphioxus nervous system and are characterized by differential expression in neurons and glia, respectively. However, in vertebrates, glycinergic neurons express GlyT2 and glia GlyT1, suggesting that the evolution of the chordate glycinergic system was accompanied by complex genetic remodeling leading to the paralog-specific inversion of gene expression. Albeit this genetic divergence between amphioxus and vertebrates, we found strong evidence for a general conservation of the role of glycinergic neurotransmission during larval swimming, allowing us to hypothesize that the neural networks controlling the rhythmic movement of chordate bodies are homologous.

Holothuroids (sea cucumbers) are one of the most ubiquitous groups of benthic animals found across diverse marine ecosystems. As echinoderms, they also occupy an important place in the evolutionary hierarchy, sitting close to vertebrates in the deuterostome clade, making them valuable multidisciplinary model organisms. Apart from being ecologically and phylogenetically important, many species are commercially exploited for luxury seafood markets. With the global rise of aquaculture and fisheries, management and protection of these valuable species relies on a better understanding of how their immune systems respond to environmental and anthropogenic stressors. Here, the cellular, hormonal and behavioral indicators of stress in the North Atlantic sea cucumberCucumaria frondosawere examined. The immediate and carry-over (post recovery) effects of a 1-hour exposure to low salinities or to emersion (at two temperatures) highlighted that morphoplasticity inC. frondosawas accompanied by shifts in all monitored indicators. From baseline levels measured in controls, densities of free coelomocytes increased, showing successions of specific cell types and subsequent coelomocyte aggregations, combined with a rise in cortisol levels. These responses mirrored increased fluctuations in cloacal opening rates, decreased force of attachment to the substrate, and enhanced movements and active buoyancy adjustment with increasingly severe stressors. The findings suggest that many systems of sea cucumbers are impacted by stresses that can be associated with harvesting and handling methods, with likely implications for the quality of the processed products. Gaining a deeper understanding of immune and hormonal responses of sea cucumbers is not only of broad ecological and evolutionary value, but also helpful for the development of sustainable fisheries and aquaculture practices, and conservation programs.

Programmed cell death (PCD) has been identified as a key process in the metamorphic transition of indirectly developing organisms such as frogs and insects. Many marine invertebrate species with indirect development and biphasic life cycles face the challenge of completing the metamorphic transition of the larval body into a juvenile when they settle into the benthic habitat. Some key characteristics stand out during this transition in comparison to frogs and insects: (1) the transition is often remarkably fast and (2) the larval body is largely abandoned and few structures transition into the juvenile stage. In sea urchins, a group with a drastic and fast metamorphosis, development and destruction of the larval body is regulated by endocrine signals. Here we provide a brief review of the basic regulatory mechanisms of PCD in animals. We then narrow our discussion to metamorphosis with a specific emphasis on sea urchins with indirect life histories and discuss the function of thyroid hormones and histamine in larval development, metamorphosis and settlement of the sea urchin Strongylocentrotus purpuratus. We were able to annotate the large majority of PCD related genes in the sea urchin S. purpuratus and ongoing studies on sea urchin metamorphosis will shed light on the regulatory architecture underlying this dramatic life history transition. While we find overwhelming evidence for hormonal regulation of PCD in animals, especially in the context of metamorphosis, the mechanisms in many marine invertebrate groups with indirect life histories requires more work. Hence, we propose that studies of PCD in animals requires functional studies in whole organisms rather than isolated cells. We predict that future work, targeting a broader array of organisms will not only help to reveal important new functions of PCD but provide a fundamentally new perspective on its use in a diversity of taxonomic, developmental, and ecological contexts.

Sulfakinin (SK)/cholecystokinin (CCK)-type neuropeptides regulate feeding and digestion in protostomes (e.g. insects) and chordates. Here, we characterised SK/CCK-type signalling for the first time in a non-chordate deuterostome – the starfish Asterias rubens (phylum Echinodermata). In this species, two neuropeptides (ArSK/CCK1, ArSK/CCK2) derived from the precursor protein ArSK/CCKP act as ligands for an SK/CCK-type receptor (ArSK/CCKR) and these peptides/proteins are expressed in the nervous system, digestive system, tube feet, and body wall. Furthermore, ArSK/CCK1 and ArSK/CCK2 cause dose-dependent contraction of cardiac stomach, tube foot, and apical muscle preparations in vitro, and injection of these neuropeptides in vivo triggers cardiac stomach retraction and inhibition of the onset of feeding in A. rubens. Thus, an evolutionarily ancient role of SK/CCK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

Sea urchins, in many instances, are collected from the wild, maintained in the laboratory aquaculture environment, and used as model animals for various scientific investigations. It has been increasingly evident that diet-driven dysbiosis of the gut microbiome could affect animal health and physiology, thereby impacting the outcome of the scientific studies. In this study, we compared the gut microbiome between naturally occurring (ENV) and formulated diet-fed laboratory aquaculture (LAB) sea urchin Lytechinus variegatus by amplicon sequencing of the V4 region of the 16S rRNA gene and bioinformatics tools. Overall, the ENV gut digesta had higher taxa richness with an abundance of Propionigenium, Photobacterium, Roseimarinus, and Flavobacteriales. In contrast, the LAB group revealed fewer taxa richness, but noticeable abundances of Arcobacter, Agarivorans, and Shewanella. However, Campylobacteraceae, primarily represented by Arcobacter spp., was commonly associated with the gut tissues of both ENV and LAB groups whereas the gut digesta had taxa from Gammaproteobacteria, particularly Vibrio spp. Similarly, the co-occurrence network displayed taxonomic organizations interconnected by Arcobacter and Vibrio as being the key taxa in gut tissues and gut digesta, respectively. Predicted functional analysis of the gut tissues microbiota of both ENV and LAB groups showed a higher trend in energy-related metabolisms, whereas amino acids, carbohydrate, and lipid metabolisms heightened in the gut digesta. This study provides an outlook of the laboratory-formulated diet-fed aquaculture L. variegatus gut microbiome and predicted metabolic profile as compared to the naturally occurring animals, which should be taken into consideration for consistency, reproducibility, and translatability of scientific studies.

In their seminal paper, Gans and Northcutt (1983) proposed that evolution of the vertebrate “New Head” was enabled by advent of the neural crest and cranial placodes(1). The neural crest is a stem cell population that arises adjacent to the forming central nervous system and contributes to important cell types including components of the peripheral nervous system, craniofacial skeleton, and elements of the cardiovascular system(2). In the past few years, the New Head hypothesis has been challenged by the discovery in invertebrate chordates of cells with some but not all characteristics of vertebrate neural crest cells(3–7). Here, we discuss recent findings regarding how neural crest cells may have evolved during the course of deuterostome evolution. The results suggest that there was progressive addition of cell types into the repertoire of neural crest derivatives throughout vertebrate evolution(8). Novel genomic tools have enabled higher resolution insight into neural crest evolution from both a cellular and gene regulatory perspective(9,10). Together, these data provide clues regarding the ancestral neural crest state and how the neural crest continues to evolve to contribute to the success of vertebrates as efficient predators.

The ability to regenerate is scattered among the metazoan tree of life. Further still, regenerative capacity varies widely within these specific organisms. Numerous organisms, all with different regenerative capabilities, have been studied at length and key similarities and disparities in how regeneration occurs have been identified. In order to get a better grasp on understanding regeneration as a whole, we must search for new models that are capable of extensive regeneration, as well as those that have been under sampled in the literature. As invertebrate deuterostomes, echinoderms fit both of these requirements. Multiple members regenerate various tissue types at all life stages, including examples of whole-body regeneration. Interrogations in two highly studied echinoderms, the sea urchin and the sea star, have provided knowledge of tissue and whole-body regeneration at various life stages. Work has begun to examine regeneration in echinoderm larvae, a potential new system for understanding regenerative mechanisms in a basal deuterostome. Here, we review the ways these two animals’ larvae have been utilized as a model of regeneration.

Background The phyla Cnidaria, Placozoa, Ctenophora, and Porifera emerged before the split of proto- and deuterostome animals, about 600 million years ago. These early metazoans are interesting, because they can give us important information on the evolution of various tissues and organs, such as eyes and the nervous system. Generally, cnidarians have simple nervous systems, which use neuropeptides for their neurotransmission, but some cnidarian medusae belonging to the class Cubozoa (box jellyfishes) have advanced image-forming eyes, probably associated with a complex innervation. Here, we describe a new transcriptome database from the cubomedusa Tripedalia cystophora. Results Based on the combined use of the Illumina and PacBio sequencing technologies, we produced a highly contiguous transcriptome database from T. cystophora. We then developed a software program to discover neuropeptide preprohormones in this database. This script enabled us to annotate seven novel T. cystophora neuropeptide preprohormone cDNAs: One coding for 19 copies of a peptide with the structure pQWLRGRFamide; one coding for six copies of a different RFamide peptide; one coding for six copies of pQPPGVWamide; one coding for eight different neuropeptide copies with the C-terminal LWamide sequence; one coding for thirteen copies of a peptide with the RPRAamide C-terminus; one coding for four copies of a peptide with the C-terminal GRYamide sequence; and one coding for seven copies of a cyclic peptide, of which the most frequent one has the sequence CTGQMCWFRamide. We could also identify orthologs of these seven preprohormones in the cubozoans Alatina alata, Carybdea xaymacana, Chironex fleckeri, and Chiropsalmus quadrumanus. Furthermore, using TBLASTN screening, we could annotate four bursicon-like glycoprotein hormone subunits, five opsins, and 52 other family-A G protein-coupled receptors (GPCRs), which also included two leucine-rich repeats containing G protein-coupled receptors (LGRs) in T. cystophora. The two LGRs are potential receptors for the glycoprotein hormones, while the other GPCRs are candidate receptors for the above-mentioned neuropeptides. Conclusions By combining Illumina and PacBio sequencing technologies, we have produced a new high-quality de novo transcriptome assembly from T. cystophora that should be a valuable resource for identifying the neuronal components that are involved in vision and other behaviors in cubomedusae. Electronic supplementary material The online version of this article (10.1186/s12864-019-5514-7) contains supplementary material, which is available to authorized users.

Background The sea urchin is a basal deuterostome that is more closely related to vertebrates than many organisms traditionally used to study neurogenesis. This phylogenetic position means that the sea urchin can provide insights into the evolution of the nervous system by helping resolve which developmental processes are deuterostome innovations, which are innovations in other clades, and which are ancestral. However, the nervous system of echinoderms is one of the least understood of all major metazoan phyla. To gain insights into echinoderm neurogenesis, spatial and temporal gene expression data are essential. Then, functional data will enable the building of a detailed gene regulatory network for neurogenesis in the sea urchin that can be compared across metazoans to resolve questions about how nervous systems evolved. Results Here, we analyze spatiotemporal gene expression during sea urchin neurogenesis for genes that have been shown to be neurogenic in one or more species. We report the expression of 21 genes expressed in areas of neurogenesis in the sea urchin embryo from blastula stage (just before neural progenitors begin their specification sequence) through pluteus larval stage (when much of the nervous system has been patterned). Among those 21 gene expression patterns, we report expression of 11 transcription factors and 2 axon guidance genes, each expressed in discrete domains in the neuroectoderm or in the endoderm. Most of these genes are expressed in and around the ciliary band. Some including the transcription factors Lv-mbx, Lv-dmrt, Lv-islet, and Lv-atbf1, the nuclear protein Lv-prohibitin, and the guidance molecule Lv-semaa are expressed in the endoderm where they are presumably involved in neurogenesis in the gut. Conclusions This study builds a foundation to study how neurons are specified and evolved by analyzing spatial and temporal gene expression during neurogenesis in a basal deuterostome. With these expression patterns, we will be able to understand what genes are required for neural development in the sea urchin. These data can be used as a starting point to (1) build a spatial gene regulatory network for sea urchin neurogenesis, (2) identify how subtypes of neurons are specified, (3) perform comparative studies with the sea urchin, protostome, and vertebrate organisms. Electronic supplementary material The online version of this article (10.1186/s13227-019-0115-8) contains supplementary material, which is available to authorized users.

BackgroundMetazoan lineages exhibit a wide range of regenerative capabilities that vary among developmental stage and tissue type. The most robust regenerative abilities are apparent in the phyla Cnidaria, Platyhelminthes, and Echinodermata, whose members are capable of whole-body regeneration (WBR). This phenomenon has been well-characterized in planarian and hydra models, but the molecular details of WBR are less established within echinoderms, or any other deuterostome system. Thus, it is not clear to what degree aspects of this regenerative ability are due to deeply conserved mechanisms.ResultsWe characterize regeneration in the larval stage of the Bat Star (Patiria miniata). Following bisection along the anterior-posterior axis, larvae progress through phases of wound healing and re-proportioning of larval tissues. The overall number of proliferating cells is reduced following bisection and we find evidence for a re-deployment of genes with known roles in embryonic axial patterning. Following axial re-specification, we observe a significant localization of proliferating cells to the wound region. Analyses of transcriptome data highlight the molecular signatures of functions that are common to regeneration, including specific signaling pathways and cell cycle controls. Notably, we find evidence for temporal conservation among orthologous genes involved in regeneration from published Platyhelminth and Cnidarian regeneration datasets.ConclusionsThese analyses show that sea star larval regeneration includes phases of wound response, axis respecification, and wound proximal proliferation. Commonalities of the overall process of regeneration, as well as gene usage between this deuterostome and other species with divergent evolutionary origins suggest a deep conservation of whole-body regeneration among the metazoa.

Background Cilia and flagella are complex cellular structures thought to have first evolved in a last ciliated eukaryotic ancestor due to the conserved 9 + 2 microtubule doublet structure of the axoneme and associated proteins. The Tektin family of coiled-coil domain containing proteins was previously identified in cilia of organisms as diverse as green algae and sea urchin. While studies have shown that some Tektins are necessary for ciliary function, there has been no comprehensive phylogenetic survey of tektin genes. To fill this gap, we sampled tektin sequences broadly among metazoan and unicellular lineages in order to determine how the tektin gene complements evolved in over 100 different extant species. Results Using Bayesian and Maximum Likelihood analyses, we have ascertained with high confidence that all metazoan tektins arose from a single ancestral tektin gene in the last common ancestor of metazoans and choanoflagellates. Gene duplications gave rise to two tektin genes in the metazoan ancestor, and a subsequent expansion to three and four tektin genes in early bilaterian ancestors. While all four tektin genes remained highly conserved in most deuterostome and spiralian species surveyed, most tektin genes in ecdysozoans are highly derived with extensive gene loss in several lineages including nematodes and some crustaceans. In addition, while tektin-1, − 2, and − 4 have remained as single copy genes in most lineages, tektin-3/5 has been duplicated independently several times, notably at the base of the spiralian, vertebrate and hymenopteran (Ecdysozoa) clades. Conclusions We provide a solid description of tektin evolution supporting one, two, three, and four ancestral tektin genes in a holozoan, metazoan, bilaterian, and nephrozoan ancestor, respectively. The isolated presence of tektin in a cryptophyte and a chlorophyte branch invokes events of horizontal gene transfer, and that the last common ciliated eukaryotic ancestor lacked a tektin gene. Reconstructing the evolutionary history of the tektin complement in each extant metazoan species enabled us to pinpoint lineage specific expansions and losses. Our analysis will help to direct future studies on Tektin function, and how gain and loss of tektin genes might have contributed to the evolution of various types of cilia and flagella. Electronic supplementary material The online version of this article (10.1186/s12862-019-1360-0) contains supplementary material, which is available to authorized users.

Apoptosis and autophagy are fundamental mechanisms of programed cell death activated during protostome and deuterostome embryonic development, contributing to the creation and remodeling of different anatomical structures. Programed cell death has been investigated at morphological and biochemical levels, but there is a lack of information concerning gene expression of death factors during deuterostome embryonic development. In this study, we analyze the expression patterns of 13 genes involved in autophagy, extrinsic and intrinsic apoptosis during blastula, gastrula, and pluteus stages of the sea urchin Paracentrotus lividus embryonic development. Results suggested the occurrence of all death mechanisms investigated, highlighting the simultaneous involvement of apoptosis and autophagy during embryonic development. In particular, gastrula was the developmental stage where the majority of death genes were highly expressed. During gastrulation apoptotic processes are fundamental for tissue remodeling, such as cavity formation and removal of inner ectodermal cells. This is the first report that identifies a panel of cell death genes in the P. lividus genome and analyzes their expression variations during ontogenesis.

Activation of Wnt/β-catenin (cWnt) signaling at the future posterior end of early bilaterian embryos is a highly conserved mechanism for establishing the anterior-posterior (AP) axis. Moreover, inhibition of cWnt at the anterior end is required for development of anterior structures in many deuterostome taxa. This phenomenon, which occurs around the time of gastrulation, has been fairly well characterized, but the significance of intracellular inhibition of cWnt signaling in cleavage-stage deuterostome embryos for normal AP patterning is less well understood. To investigate this process in an invertebrate deuterostome, we defined Axin function in early sea urchin embryos. Axin is ubiquitously expressed at relatively high levels in early embryos and functional analysis revealed that Axin suppresses posterior cell fates in anterior blastomeres by blocking ectopic cWnt activation in these cells. Structure-function analysis of sea urchin Axin demonstrated that only its GSK-3β-binding domain is required for cWnt inhibition. These observations and results in other deuterostomes suggest that Axin plays a crucial conserved role in embryonic AP patterning by preventing cWnt activation in multipotent early blastomeres, thus protecting them from assuming ectopic cell fates., Summary: Axin function is required in the early sea urchin embryo to regulate nuclear β-catenin levels and prevent ectopic cell fates in multipotent early blastomeres, and to ensure correct anterior-posterior axis patterning.

Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identified 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these revealed a highly detailed portrait of cell diversity across the larva, including the identification of 12 distinct neuronal cell types. Moreover, we corroborated co-expression of key regulatory genes previously shown to drive sea urchin gene regulatory networks, and revealed additional domains in which these regulatory networks are likely to function within the larva. Lastly, we recovered a neuronal cell type co-expressing Pdx-1 and Brn1/2/4, which had previously been shown to share similar gene expression with vertebrate pancreas. Our results extend this finding, revealing twenty transcription factors shared by this population of neurons in sea urchin and vertebrate pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we generate a draft of the Pdx-1 regulatory network in these cells, and hypothesize this network was present in an ancestral deuterostome neuron before being co-opted into the pancreas developmental lineage in vertebrates.

Recent efforts have been devoted to the link between responses to non-physical stressors and immune states in animals, mostly using human and other vertebrate models. Despite evolutionary relevance, comparatively limited work on the appraisal of predation risk and aspects of cognitive ecology and ecoimmunology has been carried out in non-chordate animals. The present study explored the capacity of holothuroid echinoderms to display an immune response to both reactive and anticipatory predatory stressors. Experimental trials and a mix of behavioural, cellular and hormonal markers were used, with a focus on coelomocytes (analogues of mammalian leukocytes), which are the main components of the echinoderm innate immunity. Findings suggest that holothuroids can not only appraise threatening cues (i.e. scent of a predator or alarm signals from injured conspecifics) but prepare themselves immunologically, presumably to cope more efficiently with potential future injuries. The responses share features with recently defined central emotional states and wane after prolonged stress in a manner akin to habituation, which are traits that have rarely been shown in non-vertebrates, and never in echinoderms. Because echinoderms sit alongside chordates in the deuterostome clade, such findings offer unique insights into the adaptive value and evolution of stress responses in animals.

Background Regeneration is the ability to re-grow body parts or tissues after trauma, and it is widespread across metazoans. Cells involved in regeneration can arise from a pool of undifferentiated proliferative cells or be recruited from pre-existing differentiated tissues. Both mechanisms have been described in different phyla; however, the cellular and molecular mechanisms employed by different animals to restore lost tissues as well as the source of cells involved in regeneration remain largely unknown. Echinoderms are a clade of deuterostome invertebrates that show striking larval and adult regenerative abilities in all extant classes. Here, we use the brittle star Amphiura filiformis to investigate the origin and differentiation of cells involved in skeletal regeneration using a combination of microscopy techniques and molecular markers. Results Our ultrastructural analyses at different regenerative stages identify a population of morphologically undifferentiated cells which appear in close contact with the proliferating epithelium of the regenerating aboral coelomic cavity. These cells express skeletogenic marker genes, such as the transcription factor alx1 and the differentiation genes c-lectin and msp130L, and display a gradient of morphological differentiation from the aboral coelomic cavity towards the epidermis. Cells closer to the epidermis, which are in contact with developing spicules, have the morphology of mature skeletal cells (sclerocytes), and express several skeletogenic transcription factors and differentiation genes. Moreover, as regeneration progresses, sclerocytes show a different combinatorial expression of genes in various skeletal elements. Conclusions We hypothesize that sclerocyte precursors originate from the epithelium of the proliferating aboral coelomic cavity. As these cells migrate towards the epidermis, they differentiate and start secreting spicules. Moreover, our study shows that molecular and cellular processes involved in skeletal regeneration resemble those used during skeletal development, hinting at a possible conservation of developmental programmes during adult regeneration. Finally, we highlight that many genes involved in echinoderm skeletogenesis also play a role in vertebrate skeleton formation, suggesting a possible common origin of the deuterostome endoskeleton pathway.

International audience; The echinoderms are a phylum of marine deuterostomes characterized by the pentaradial (five fold) symmetry of their adult bodies. Due to this unusual body plan, adult echinoderms have long been excluded from comparative analyses aimed at understanding the origin and evolution of deuterostome nervous systems. Here, we investigated the neural anatomy of early juveniles of representatives of three of the five echinoderm classes: the echinoid Paracentrotus lividus, the asteroid Patiria miniata, and the holothuroid Parastichopus parvimensis. Using whole mount immunohistochemistry and confocal microscopy, we found that the nervous system of echinoid early juveniles is composed of three main structures: a basiepidermal nerve plexus, five radial nerve cords connected by a circumoral nerve ring, and peripheral nerves innervating the appendages. Our whole mount preparations further allowed us to obtain thorough descriptions of these structures and of several innervation patterns, in particular at the level of the appendages. Detailed comparisons of the echinoid juvenile nervous system with those of asteroid and holothuroid juveniles moreover supported a general conservation of the main neural structures in all three species, including at the level of the appendages. Our results support the previously proposed hypotheses for the existence of two neural units in echinoderms: one consisting of the basiepidermal nerve plexus to process sensory stimuli locally and one composed of the radial nerve cords and the peripheral nerves constituting a centralized control system. This study provides the basis for more in‐depth comparisons of the echinoderm adult nervous system with those of other animals, in particular hemichordates and chordates, to address the long‐standing controversies about deuterostome nervous system evolution.

Chemosensation is a critical signalling process in animals and especially important in sea cucumbers, a group of ecologically and economically important marine echinoderms (class Holothuroidea), which lack audio and visual organs and rely on chemical sensing for survival, feeding and reproduction. The ionotropic receptors are a recently identified family of chemosensory receptors in insects and other protostomes, related to the ionotropic glutamate receptor family (iGluR), a large family of membrane receptors in metazoan. Here we characterize the echinoderm iGluR subunits and consider their possible role in chemical communication in sea cucumbers. Sequence similarity searches revealed that sea cucumbers have in general a higher number of iGluR subunits when compared to other echinoderms. Phylogenetic analysis and sequence comparisons revealed GluH as a specific iGluR subfamily present in all echinoderms. Homologues of the vertebrate GluA (aka alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, AMPA), GluK (aka kainate) and GluD (aka delta) were also identified. The GluN (aka N-methyl-d-aspartate, NMDA) as well as the invertebrate deuterostome subfamily GluF (aka phi) are absent in echinoderms. The echinoderm GluH subfamily shares conserved structural protein organization with vertebrate iGluRs and the ligand binding domain (LBD) is the most conserved region; genome analysis indicates evolution via lineage and species-specific tandem gene duplications. GluH genes (named Grih) are the most highly expressed iGluRs subunit genes in tissues in the sea cucumber Holothuria arguinesis, with Griha1, Griha2 and Griha5 exclusively expressed in tentacles, making them candidates to have a chemosensory role in this species. The multiple GluH subunits may provide alternative receptor assembly combinations, thus expanding the functional possibilities and widening the range of compounds detected during aggregation and spawning in echinoderms. info:eu-repo/semantics/publishedVersion

Background: Whether position Xenacoelomorpha as an early branch of Bilateria (Protostomes + Deuterostomes) has been intensely debated during last several decades. Considering Darwin’s “tree of life”, with the “Phylogenetic Species Concept”, we choose mitochondrial genome as the subject to predict phylogenetic position of Xenacoelomorpha, by genes genealogy. Results: Herein, we sequence Heterochaerus australis’s mitochondrial genome and infer intrinsic relationships of Metazoan with Xenacoelomorpha. The optimal tree under the popular maximum likelihood (ML) and Bayesian phylogenetic reconstructions are consensus with each other being strongly supported. The relationship between Chordates, Ambulacrarians and Xenoturbella/Acoelomorph is resolved. To avoid previous query about alignment process, the datasets are alignmented and trimmed automatically. Reducing taxon or cutting outgroups can not affect the relationship between Xenacoelomorpha and other Metazoan. Meanwhile, analysis using CAT model and Dayhoff groups also supporting the prediction made by mtZOA, relaxing the restriction of alignment criteria ( MAFFT, strategy G–ins–1, BLOSUM 62, 45, 30 ) introducing potential misleading signals can not challenge the tree topology indicating our auto-alignmented mitochondrial dataset is not artificially restricted one. Conclusions: Finally, a repeatable prediction of the genes genealogy with reliable statistical support places Xenacoelomorpha as a basal Deuterostome. Let's enjoy the elegent metapher about Tree of LIfe—— "As buds give rise by growth to fresh buds, and these, if vigorous, branch out and overtop on all sides many a feebler branch, so by generation we believe it has been with the great Tree of Life, which fills with its dead and broken branches the crust of the earth, and covers the surface with its ever branching and beautiful ramifications". (Darwin 1964 [1859], p. 130).

Cholecystokinin (CCK) / sulfakinin (SK)-type neuropeptides regulate feeding and digestion in chordates and protostomes (e.g. insects). Here we characterised CCK/SK-type signalling for the first time in a non-chordate deuterostome - the starfishAsterias rubens(phylum Echinodermata). In this species, two neuropeptides (ArCCK1, ArCCK2) derived from the precursor protein ArCCKP act as ligands for a CCK/SK-type receptor (ArCCKR) and are expressed in the nervous system, digestive system, tube feet and body wall. Furthermore, ArCCK1 and ArCCK2 cause dose-dependent contraction of cardiac stomach, tube foot and body wall apical muscle preparationsin vitroand injection of these neuropeptidesin vivotriggers cardiac stomach retraction and inhibition of the onset of feeding inA. rubens. Thus, an evolutionarily ancient role of CCK/SK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

Background How vascular systems and their respiratory pigments evolved is still debated. While many animals present a vascular system, hemoglobin exists as a blood pigment only in a few groups (vertebrates, annelids, a few arthropod and mollusk species). Hemoglobins are formed of globin sub-units, belonging to multigene families, in various multimeric assemblages. It was so far unclear whether hemoglobin families from different bilaterian groups had a common origin. Results To unravel globin evolution in bilaterians, we studied the marine annelid Platynereis dumerilii, a species with a slow evolving genome. Platynereis exhibits a closed vascular system filled with extracellular hemoglobin. Platynereis genome and transcriptomes reveal a family of 19 globins, nine of which are predicted to be extracellular. Extracellular globins are produced by specialized cells lining the vessels of the segmental appendages of the worm, serving as gills, and thus likely participate in the assembly of a previously characterized annelid-specific giant hemoglobin. Extracellular globin mRNAs are absent in smaller juveniles, accumulate considerably in growing and more active worms and peak in swarming adults, as the need for O2 culminates. Next, we conducted a metazoan-wide phylogenetic analysis of globins using data from complete genomes. We establish that five globin genes (stem globins) were present in the last common ancestor of bilaterians. Based on these results, we propose a new nomenclature of globins, with five clades. All five ancestral stem-globin clades are retained in some spiralians, while some clades disappeared early in deuterostome and ecdysozoan evolution. All known bilaterian blood globin families are grouped in a single clade (clade I) together with intracellular globins of bilaterians devoid of red blood. Conclusions We uncover a complex “pre-blood” evolution of globins, with an early gene radiation in ancestral bilaterians. Circulating hemoglobins in various bilaterian groups evolved convergently, presumably in correlation with animal size and activity. However, all hemoglobins derive from a clade I globin, or cytoglobin, probably involved in intracellular O2 transit and regulation. The annelid Platynereis is remarkable in having a large family of extracellular blood globins, while retaining all clades of ancestral bilaterian globins.

Members of thealxgene family encode transcription factors that contain a highly conserved Paired-class, DNA-binding homeodomain, and a C-terminal OAR/Aristaless domain. Phylogenetic and comparative genomic studies have revealed complex patterns ofalxgene duplications during deuterostome evolution. Remarkably,alxgenes have been implicated in skeletogenesis in both echinoderms and vertebrates. In this review, we provide an overview of current knowledge concerningalxgenes in deuterostomes. We highlight their evolutionarily conserved role in skeletogenesis and draw parallels and distinctions between the skeletogenic gene regulatory circuitries of diverse groups within the superphylum.

The molecular mechanisms underlying development of the pentameral body of adult echinoderms are poorly understood but are important to solve with respect to evolution of a unique body plan that contrasts with the bilateral body plan of other deuterostomes. As Nodal and BMP2/4 signalling is involved in axis formation in larvae and development of the echinoderm body plan, we used the developmental transcriptome generated for the asterinid seastar Parvulastra exigua to investigate the temporal expression patterns of Nodal and BMP2/4 genes from the embryo and across metamorphosis to the juvenile. For echinoderms, the Asteroidea represents the basal-type body architecture with a distinct (separated) ray structure. Parvulastra exigua has lecithotrophic development forming the juvenile soon after gastrulation providing ready access to the developing adult stage. We identified 39 genes associated with the Nodal and BMP2/4 network in the P. exigua developmental transcriptome. Clustering analysis of these genes resulted in 6 clusters with similar temporal expression patterns across development. A co-expression analysis revealed genes that have similar expression profiles as Nodal and BMP2/4. These results indicated genes that may have a regulatory relationship in patterning morphogenesis of the juvenile seastar. Developmental RNA-seq analyses of Parvulastra exigua show changes in Nodal and BMP2/4 signalling genes across the metamorphic transition. We provide the foundation for detailed analyses of this cascade in the evolution of the unusual pentameral echinoderm body and its deuterostome affinities.

Although Hox genes encode for conserved transcription factors (TFs), they are further divided into anterior, central and posterior groups based on their DNA-binding domain similarity. The posterior Hox group expanded in the deuterostome clade and patterns caudal and distal structures. We aimed to address how similar Hox TFs diverge to induce different positional identities. We studied Hox TF DNA-binding and regulatory activity during an in vitro motor neuron differentiation system that recapitulates embryonic development. We found diversity in the genomic binding profiles of different Hox TFs, even among the posterior group paralogs that share similar DNA-binding domains. These differences in genomic binding were explained by differing abilities to bind to previously inaccessible sites. For example, the posterior group HOXC9 had a greater ability to bind occluded sites than the posterior HOXC10, producing different binding patterns and driving differential gene expression programs. From these results, we propose that the differential abilities of posterior Hox TFs to bind to previously inaccessible chromatin drive patterning diversification. This article has an associated ‘The people behind the papers’ interview., Highlighted Article: Analyses of Hox transcription factor (TF) DNA-binding and regulatory activity demonstrates that the ability of mammalian posterior Hox TFs to bind to previously inaccessible chromatin is the predominant force driving their patterning diversification.

Somatostatin (SS) and allatostatin-C (ASTC) are structurally and evolutionarily related neuropeptides that act as inhibitory regulators of physiological processes in mammals and insects, respectively. Here, we report the first molecular and functional characterization of SS/ASTC-type signalling in a deuterostome invertebrate—the starfish Asterias rubens (phylum Echinodermata). Two SS/ASTC-type precursors were identified in A. rubens (ArSSP1 and ArSSP2) and the structures of neuropeptides derived from these proteins (ArSS1 and ArSS2) were analysed using mass spectrometry. Pharmacological characterization of three cloned A. rubens SS/ASTC-type receptors (ArSSR1–3) revealed that ArSS2, but not ArSS1, acts as a ligand for all three receptors. Analysis of ArSS2 expression in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed stained cells/fibres in the central nervous system, the digestive system (e.g. cardiac stomach) and the body wall and its appendages (e.g. tube feet). Furthermore, in vitro pharmacological tests revealed that ArSS2 causes dose-dependent relaxation of tube foot and cardiac stomach preparations, while injection of ArSS2 in vivo causes partial eversion of the cardiac stomach. Our findings provide new insights into the molecular evolution of SS/ASTC-type signalling in the animal kingdom and reveal an ancient role of SS-type neuropeptides as inhibitory regulators of muscle contractility.

The ecdysozoans are the most diverse animal group on Earth1, 2. Molecular clock studies indicate that the ecdysozoans may have diverged and diversified in the Ediacaran Period3, 4, but unambiguous ecdysozoan fossils first appear in the earliest Cambrian and are limited to cycloneuralians5–7. Here we report new material of the early Cambrian microscopic animalSaccorhytus coronarius, which was previously interpreted as a deuterostome8.Saccorhytus coronariusis reconstructed as a millimetric and ellipsoidal meiobenthic animal with a spinose armor and an anterior mouth but no anus. Purported pharyngeal gills in support of the deuterostome hypothesis8are shown to be taphonomic artifacts. Phylogenetic analyses indicate thatSaccorhytus coronariusbelongs to the total-group Ecdysozoa, highlighting the morphological and ecological diversity of early Cambrian ecdysozoans.

The deuterostome animal groups (Chordata and Xenambulacraria) may not be each other’s closest relatives., The bilaterally symmetric animals (Bilateria) are considered to comprise two monophyletic groups, Protostomia (Ecdysozoa and the Lophotrochozoa) and Deuterostomia (Chordata and the Xenambulacraria). Recent molecular phylogenetic studies have not consistently supported deuterostome monophyly. Here, we compare support for Protostomia and Deuterostomia using multiple, independent phylogenomic datasets. As expected, Protostomia is always strongly supported, especially by longer and higher-quality genes. Support for Deuterostomia, however, is always equivocal and barely higher than support for paraphyletic alternatives. Conditions that cause tree reconstruction errors—inadequate models, short internal branches, faster evolving genes, and unequal branch lengths—coincide with support for monophyletic deuterostomes. Simulation experiments show that support for Deuterostomia could be explained by systematic error. The branch between bilaterian and deuterostome common ancestors is, at best, very short, supporting the idea that the bilaterian ancestor may have been deuterostome-like. Our findings have important implications for the understanding of early animal evolution.

The Cambrian radiation represents a key time period in the history of life. Here, we add to the mounting evidence accumulating on the nature of deuterostomes from this time period through description of a new species of stalked deuterostome, Herpetogaster haiyanensis nov. sp., from the lower Cambrian (series 2, stage 3) Chengjiang biota of China. This represents the first occurrence of the genus in Gondwana, the first juvenile specimen, and the oldest specimens to date. Herpetogaster haiyanensis nov. sp. differs from H. collinsi Caron et al. (2010) in having a stolon that is separated into an outer and inner layer, the segmentation of the body and in the shape and number of branches of the tentacles. The new species reiterates earlier suggestions of deuterostome affinities of the genus―it appears closely related to Phlogites and then successively more distantly related to Cotyledon and Eldonia―and may have fed on hyolithids.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a well-conserved neuropeptide characteristic of vertebrates. This pluripotent hypothalamic neuropeptide regulates neurotransmitter release, intestinal motility, metabolism, cell division/differentiation, and immunity. In vertebrates, PACAP has a specific receptor (PAC1) but it can also activate the Vasoactive Intestinal Peptide receptors (VPAC1 and VPAC2). The evolution of the vertebrate PACAP ligand - receptor pair has been well-described. In contrast, the situation in invertebrates is much less clear. The PACAP ligand - receptor pair in invertebrates has mainly been studied using heterologous antibodies raised against mammalian peptides. A few partial PACAP cDNA clones sharing >87% aa identity with vertebrate PACAP have been isolated from a cnidarian, several protostomes and tunicates but no gene has been reported. Moreover, current evolutionary models of the peptide and receptors using molecular data from phylogenetically distinct invertebrate species (mostly nematodes and arthropods) suggests the PACAP ligand and receptors are exclusive to vertebrate genomes. A basal deuterostome, the cephalochordate amphioxus (Branchiostoma floridae), is the only invertebrate in which elements of a PACAP-like system exists but the peptides and receptor share relatively low sequence conservation with the vertebrate homolog system and are a hybrid with the vertebrate glucagon system. In this study, the evolution of the PACAP system is revisited taking advantage of the burgeoning sequence data (genome and transcriptomes) available for invertebrates to uncover clues about when it first appeared. The results suggest that elements of the PACAP system are absent from protozoans, non-bilaterians, and protostomes and they only emerged after the protostome-deuterostome divergence. PACAP and its receptors appeared in vertebrate genomes and they probably shared a common ancestral origin with the cephalochordate PACAP/GCG-like system which after the genome tetraploidization events that preceded the vertebrate radiation generated the PACAP ligand and receptor pair and also the other members of the Secretin family peptides and their receptors. FCT: UIDB/04326/2020; CRESC Algarve 2020 and COMPETE 2020: EMBRC.PT ALG-01-0145-FEDER-022121 info:eu-repo/semantics/publishedVersion