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Cambrian vetulicolians have mosaic characteristics of both deuterostomes and protostomes, which has important implications for the origin and early evolution of the Deuterostomia. They are intriguing in their bizarre body plan with a series of pharyngeal gill slits. The anterior section is characterized by five pairs of gill pouches, while the paddle‐like posterior part is composed of seven or more segments. Although the overall external morphology and some internal anatomies of vetulicolians, such as pharyngeal cavity, gill system and alimentary canal, have largely been clarified, mysteries remain regarding some other internal structures, and their functional interpretations are highly controversial. In this study we identify an important but hitherto unrecognized feature: a sub‐rounded, wrinkled structure located ventrally at the posterior section (or 'tail') of weakly sclerotized members of vetulicolians, including Xidazoon stephanus and Didazoon haoae, from the early Cambrian Chengjiang biota. The enigmatic structure consistently appears at or between the third and fourth segments of the posterior section. We show that the new structure is an internal organ in the body cavity of vetulicolians and infer that it might have functioned for reproduction, excretion or digestion. The finding of this enigmatic structure from X. stephanus and D. haoae enriches our understanding of vetulicolians and might facilitate further exploration of the anatomy and physiology of early deuterostomes. [ABSTRACT FROM AUTHOR]

Evolutionary perspectives on the deployment of immune factors following infection have been shaped by studies on a limited number of biomedical model systems with a heavy emphasis on vertebrate species. Although their contributions to contemporary immunology cannot be understated, a broader phylogenetic perspective is needed to understand the evolution of immune systems across Metazoa. In our study, we leverage differential gene expression analyses to identify genes implicated in the antiviral immune response of the acorn worm hemichordate, Saccoglossus kowalevskii , and place them in the context of immunity evolution within deuterostomes—the animal clade composed of chordates, hemichordates, and echinoderms. Following acute exposure to the synthetic viral double-stranded RNA analog, poly(I:C), we show that S. kowalevskii responds by regulating the transcription of genes associated with canonical innate immunity signaling pathways (e.g. nuclear factor κB and interferon regulatory factor signaling) and metabolic processes (e.g. lipid metabolism), as well as many genes without clear evidence of orthology with those of model species. Aggregated across all experimental time point contrasts, we identify 423 genes that are differentially expressed in response to poly(I:C). We also identify 147 genes with altered temporal patterns of expression in response to immune challenge. By characterizing the molecular toolkit involved in hemichordate antiviral immunity, our findings provide vital evolutionary context for understanding the origins of immune systems within Deuterostomia. [ABSTRACT FROM AUTHOR]

Background: Corticotropin-releasing hormone (CRH) mediates physiological responses to stressors in mammals by triggering pituitary secretion of adrenocorticotropic hormone, which stimulates adrenal release of cortisol. CRH belongs to a family of related neuropeptides that include sauvagine, urotensin-I, and urocortins in vertebrates and the diuretic hormone DH44 in insects, indicating that the evolutionary origin of this neuropeptide family can be traced to the common ancestor of the Bilateria. However, little is known about CRH-type neuropeptides in deuterostome invertebrates. Methods: Here, we used mass spectrometry, mRNA in situ hybridization, and immunohistochemistry to investigate the structure and expression of a CRH-type neuropeptide (ArCRH) in the starfish Asterias rubens (phylum Echinodermata). Results: ArCRH is a 40-residue peptide with N-terminal pyroglutamylation and C-terminal amidation, and it has a widespread pattern of expression in A. rubens. In the central nervous system comprising the circumoral nerve ring and 5 radial nerve cords, ArCRH-expressing cells and fibres were revealed in both the ectoneural region and the hyponeural region, which contains the cell bodies of motoneurons. Accordingly, ArCRH immunoreactivity was detected in innervation of the ampulla and podium of locomotory organs (tube feet), and ArCRH is the first neuropeptide to be identified as a marker for nerve fibres located in the muscle layer of these organs. ArCRH immunoreactivity was also revealed in protractile organs that mediate gas exchange (papulae), the apical muscle, and the digestive system. Conclusions: Our findings provide the first insights into CRH-type neuropeptide expression and function in the unique context of the pentaradially symmetrical body plan of an echinoderm. [ABSTRACT FROM AUTHOR]

The sesquiterpenoid hormone juvenile hormone (JH) controls development, reproduction, and metamorphosis in insects, and has long been thought to be confined to the Insecta. While it remains true that juvenile hormone is specifically synthesized in insects, other types or forms of sesquiterpenoids have also been discovered in distantly related animals, such as the jellyfish. Here, we combine the latest literature and annotate the sesquiterpenoid biosynthetic pathway genes in different animal genomes. We hypothesize that the sesquiterpenoid hormonal system is an ancestral system established in an animal ancestor and remains widespread in many animals. Different animal lineages have adapted different enzymatic routes from a common pathway, with cnidarians producing farnesoic acid (FA); non-insect protostomes and non-vertebrate deuterostomes such as cephalochordate and echinoderm synthesizing FA and methyl farnesoate (MF); and insects producing FA, MF, and JH. Our hypothesis revolutionizes the current view on the sesquiterpenoids in the metazoans, and forms a foundation for a re-investigation of the roles of this important and yet neglected type of hormone in different animals. [ABSTRACT FROM AUTHOR]

Gill function in gas exchange and ion regulation has played key roles in the evolution of fishes. In this review, we summarize data from the fields of palaeontology, developmental biology and comparative physiology for when and how the gills first acquired these functions. Data from across disciplines strongly supports a stem vertebrate origin for gas exchange structures and function at the gills with the emergence of larger, more active fishes. However, the recent discovery of putative ionocytes in extant cephalochordates and hemichordates suggests that ion regulation at gills might have originated much earlier than gas exchange, perhaps in the ciliated pharyngeal arches in the last common ancestor of deuterostomes. We hypothesize that the ancestral form of ion regulation served a filter-feeding function in the ciliated pharyngeal arches, and was later coopted in vertebrates to regulate extracellular ion and acid–base balance. We propose that future research should explore ionocyte homology and function across extant deuterostomes to test this hypothesis and others in order to determine the ancestral origins of ion regulation in fish gills. [ABSTRACT FROM AUTHOR]

Ikaros family proteins (Ikaros, Helios, Aiolos, Eos) are zinc finger transcription factors essential for the development and function of the adaptive immune system. They also control developmental events in neurons and other cell types, suggesting that they possess crucial functions across disparate cell types. These functions are likely shared among the organisms in which these factors exist, and it is thus important to obtain a view of their distribution and conservation across organisms. How this family evolved remains poorly understood. Here we mined protein, mRNA and DNA databases to identify proteins with DNA-binding domains homologous to that of Ikaros. We show that Ikaros-related proteins exist in organisms from all four deuterostome phyla (chordates, echinoderms, hemichordates, xenacoelomorpha), but not in more distant groups. While most non-vertebrates have a single family member, this family grew to six members in the acoel worm Hofstenia miamia , three in jawless and four in jawed vertebrates. Most residues involved in DNA contact from zinc fingers 2 to 4 were identical across the Ikaros family, suggesting conserved mechanisms for target sequence recognition. Further, we identified a novel KRKxxxPxK/R motif that inhibits DNA binding in vitro which was conserved across the deuterostome phyla. We also identified a EψψxxxψM(D/E)QAIxxAIxYLGA(D/E)xL motif conserved among human Ikaros, Aiolos, Helios and subsets of chordate proteins, and motifs that are specific to subsets of vertebrate family members. Some of these motifs are targets of mutations in human patients. Finally we show that the atypical family member Pegasus emerged only in vertebrates, which is consistent with its function in bone. Our data provide a novel evolutionary perspective for Ikaros family proteins and suggest that they have conserved regulatory functions across deuterostomes. • Proteins with DNA binding and dimerization domains homologous to those of human Ikaros exist in all deuterostome phyla. • We identify six novel conserved motifs in human Ikaros family proteins, that emerged at various junctures during evolution. • The Ikaros/Helios/Aiolos/Eos 4-member family is specific to jawed vertebrates. • Ikaros, Helios, Aiolos and Eos each have acquired a specific identity through gain and loss of conserved motifs. • The atypical family member Pegasus is specific to vertebrates. [ABSTRACT FROM AUTHOR]

Abstract Background Proteomic studies of skeletal proteins have revealed large, complex mixtures of proteins occluded within the mineral. Many skeletal proteomes contain rapidly evolving proteins with repetitive domains, further complicating our understanding. In echinoderms, proteomic analysis of the skeletal proteomes of mineralized tissues of the sea urchin Strongylocentrotus purpuratus prominently featured spicule matrix proteins with repetitive sequences linked to a C-type lectin domain. A comparative study of the brittle star Ophiocoma wendtii skeletal proteome revealed an order of magnitude fewer proteins containing C-type lectin domains. A number of other proteins conserved in the skeletons of the two groups were identified. Here we report the complete skeletal proteome of the sea star Patiria miniata and compare it to that of the other echinoderm groups. Results We have identified eighty-five proteins in the P. miniata skeletal proteome. Forty-two percent of the proteins were determined to be homologous to proteins found in the S. purpuratus skeletal proteomes. An additional 34 % were from similar functional classes as proteins in the urchin proteomes. Thirteen percent of the P. miniata proteins had homologues in the O. wendtii skeletal proteome with an additional 29% showing similarity to brittle star skeletal proteins. The P. miniata skeletal proteome did not contain any proteins with C-lectin domains or with acidic repetitive regions similar to the sea urchin or brittle star spicule matrix proteins. MSP130 proteins were also not found. We did identify a number of proteins homologous between the three groups. Some of the highly conserved proteins found in echinoderm skeletons have also been identified in vertebrate skeletons. Conclusions The presence of proteins conserved in the skeleton in three different echinoderm groups indicates these proteins are important in skeleton formation. That a number of these proteins are involved in skeleton formation in vertebrates suggests a common origin for some of the fundamental processes co-opted for skeleton formation in deuterostomes. The proteins we identify suggest transport of proteins and calcium via endosomes was co-opted to this function in a convergent fashion. Our data also indicate that modifications to the process of skeleton formation can occur through independent co-option of proteins following species divergence as well as through domain shuffling.

Arm loss through a separation at a specialized autotomy plane in echinoderms is inextricably linked to regeneration, but the link between these phenomena is poorly explored. We investigated nervous system regeneration post-autotomy in the asteriid seastar Coscinasterias muricata, focusing on the reorganization of the radial nerve cord (RNC) into the ectoneural neuroepithelium and neuropile, and the hyponeural region, using antibodies to the seastar-specific neuropeptide SALMFamide-1 (S1). Parallel changes in the associated haemal and coelomic vessels were also examined. A new arm bud appeared in 3–5 days with regeneration over three weeks. At the nerve stump and in the RNC immediately behind, the haemal sinus/hyponeural coelomic compartments enlarged into a hypertrophied space filled with migratory cells that appear to be involved in wound healing and regeneration. The haemal and coelomic compartments provided a conduit for these cells to gain rapid access to the regeneration site. An increase in the number of glia-like cells indicates the importance of these cells in regeneration. Proximal to the autotomy plane, the original RNC exhibited Wallerian-type degeneration, as seen in disorganized axons and enlarged S1-positive varicosities. The imperative to regrow lost arms quickly is reflected in the efficiency of regeneration from the autotomy plane facilitated by the rapid appearance of progenitor-like migratory cells. In parallel to its specialization for defensive arm detachment, the autotomy plane appears to be adapted to promote regeneration. This highlights the importance of examining autotomy-induced regeneration in seastars as a model system to study nervous system regeneration in deuterostomes and the mechanisms involved with the massive migration of stem-like cells to facilitate rapid recovery. [ABSTRACT FROM AUTHOR]

Abstract: Molluscan pedal peptides (PPs) and arthropod orcokinins (OKs) are prototypes of a family of neuropeptides that have been identified in several phyla. Recently, starfish myorelaxant peptide (SMP) was identified as a PP/OK‐type neuropeptide in the starfish Patiria pectinifera (phylum Echinodermata). Furthermore, analysis of transcriptome sequence data from the starfish Asterias rubens revealed two PP/OK‐type precursors: an SMP‐type precursor (A. rubens PP‐like neuropeptide precursor 1; ArPPLNP1) and a second precursor (ArPPLNP2). We reported previously a detailed analysis of ArPPLNP1 expression in A. rubens and here we report the first functional characterization ArPPLNP2‐derived neuropeptides. Sequencing of a cDNA encoding ArPPLNP2 revealed that it comprises eleven related neuropeptides (ArPPLN2a‐k), the structures of several of which were confirmed using mass spectrometry. Analysis of the expression of ArPPLNP2 and neuropeptides derived from this precursor using mRNA in situ hybridization and immunohistochemistry revealed a widespread distribution, including expression in radial nerve cords, circumoral nerve ring, digestive system, tube feet and innervation of interossicular muscles. In vitro pharmacology revealed that the ArPPLNP2‐derived neuropeptide ArPPLN2h has no effect on the contractility of tube feet or the body wall‐associated apical muscle, contrasting with the relaxing effect of ArPPLN1b (ArSMP) on these preparations. ArPPLN2h does, however, cause dose‐dependent relaxation of cardiac stomach preparations, with greater potency/efficacy than ArPPLN1b and with similar potency/efficacy to the SALMFamide neuropeptide S2. In conclusion, there are similarities in the expression patterns of ArPPLNP1 and ArPPLNP2 but our data also indicate specialization in the roles of neuropeptides derived from these two PP/OK‐type precursors in starfish. [ABSTRACT FROM AUTHOR]

Background: Photoreception-associated genes of the Pax-Six-Eya-Dach network (PSEDN) are deployed for many roles in addition to photoreception development. In this first study of PSEDN genes during development of the pentameral body in sea urchins, we investigated their spatial expression in Heliocidaris erythrogramma. Results: Expression of PSEDN genes in the hydrocoele of early ( Dach, Eya, Six1/2) and/or late ( Pax6, Six3/6) larvae, and the five hydrocoele lobes, the first morphological expression of pentamery, supports a role in body plan development. Pax6, Six1/2, and Six3/6 were localized to the primary and/or secondary podia and putative sensory/neuronal cells. Six1/2 and Six3/6 were expressed in the neuropil region in the terminal disc of the podia. Dach was localized to spines. Sequential up-regulation of gene expression as new podia and spines formed was evident. Rhabdomeric opsin and pax6 protein were localized to cells in the primary podia and spines. Conclusions: Our results support roles for PSEDN genes in development of the pentameral body plan, contributing to our understanding of how the most unusual body plan in the Bilateria may have evolved. Development of sensory cells within the Pax-Six expression field is consistent with the role of these genes in sensory cell development in diverse species. Developmental Dynamics 247:239-249, 2018. © 2017 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Pedal peptide (PP) and orcokinin (OK) are related neuropeptides that were discovered in protostomian invertebrates (mollusks, arthropods). However, analysis of genome/transcriptome sequence data has revealed that PP/OK-type neuropeptides also occur in a deuterostomian phylum-the echinoderms. Furthermore, a PP/OK-type neuropeptide (starfish myorelaxant peptide, SMP) was recently identified as a muscle relaxant in the starfish Patiria pectinifera. Here mass spectrometry was used to identify five neuropeptides (ArPPLN1a-e) derived from the SMP precursor (PP-like neuropeptide precursor 1; ArPPLNP1) in the starfish Asterias rubens. Analysis of the expression of ArPPLNP1 and neuropeptides derived from this precursor in A. rubens using mRNA in situ hybridization and immunohistochemistry revealed a widespread pattern of expression, with labeled cells and/or processes present in the radial nerve cords, circumoral nerve ring, digestive system (e.g., cardiac stomach) and body wall-associated muscles (e.g., apical muscle) and appendages (e.g., tube feet and papulae). Furthermore, our data provide the first evidence that neuropeptides are present in the lateral motor nerves and in nerve processes innervating interossicular muscles. In vitro pharmacological tests with SMP (ArPPLN1b) revealed that it causes dose-dependent relaxation of apical muscle, tube foot and cardiac stomach preparations from A. rubens. Collectively, these anatomical and pharmacological data indicate that neuropeptides derived from ArPPLNP1 act as inhibitory neuromuscular transmitters in starfish, which contrasts with the myoexcitatory actions of PP/OK-type neuropeptides in protostomian invertebrates. Thus, the divergence of deuterostomes and protostomes may have been accompanied by an inhibitory-excitatory transition in the roles of PP/OK-type neuropeptides as regulators of muscle activity. [ABSTRACT FROM AUTHOR]

Background: Proteomic studies of skeletal proteins have revealed large, complex mixtures of proteins occluded within the mineral. Many skeletal proteomes contain rapidly evolving proteins with repetitive domains, further complicating our understanding. In echinoderms, proteomic analysis of the skeletal proteomes of mineralized tissues of the sea urchin Strongylocentrotus purpuratus prominently featured spicule matrix proteins with repetitive sequences linked to a C-type lectin domain. A comparative study of the brittle star Ophiocoma wendtii skeletal proteome revealed an order of magnitude fewer proteins containing C-type lectin domains. A number of other proteins conserved in the skeletons of the two groups were identified. Here we report the complete skeletal proteome of the sea star Patiria miniata and compare it to that of the other echinoderm groups. Results: We have identified eighty-five proteins in the P. miniata skeletal proteome. Forty-two percent of the proteins were determined to be homologous to proteins found in the S. purpuratus skeletal proteomes. An additional 34% were from similar functional classes as proteins in the urchin proteomes. Thirteen percent of the P. miniata proteins had homologues in the O. wendtii skeletal proteome with an additional 29% showing similarity to brittle star skeletal proteins. The P. miniata skeletal proteome did not contain any proteins with C-lectin domains or with acidic repetitive regions similar to the sea urchin or brittle star spicule matrix proteins. MSP130 proteins were also not found. We did identify a number of proteins homologous between the three groups. Some of the highly conserved proteins found in echinoderm skeletons have also been identified in vertebrate skeletons. Conclusions: The presence of proteins conserved in the skeleton in three different echinoderm groups indicates these proteins are important in skeleton formation. That a number of these proteins are involved in skeleton formation in vertebrates suggests a common origin for some of the fundamental processes co-opted for skeleton formation in deuterostomes. The proteins we identify suggest transport of proteins and calcium via endosomes was co-opted to this function in a convergent fashion. Our data also indicate that modifications to the process of skeleton formation can occur through independent co-option of proteins following species divergence as well as through domain shuffling. [ABSTRACT FROM AUTHOR]

Background: The molecular mechanisms underlying the development of the unusual echinoderm pentameral body plan and their likeness to mechanisms underlying the development of the bilateral plans of other deuterostomes are of interest in tracing body plan evolution. In this first study of the spatial expression of genes associated with Nodal and BMP2/4 signalling during the transition to pentamery in sea urchins, we investigate Heliocidaris erythrogramma, a species that provides access to the developing adult rudiment within days of fertilization. Results: BMP2/4, and the putative downstream genes, Six1/2, Eya, Tbx2/3 and Msx were expressed in the earliest morphological manifestation of pentamery during development, the five hydrocoele lobes. The formation of the vestibular ectoderm, the specialized region overlying the left coelom that forms adult ectoderm, involved the expression of putative Nodal target genes Chordin, Gsc and BMP2/4 and putative BMP2/4 target genes Dlx, Msx and Tbx. The expression of Nodal, Lefty and Pitx2 in the right ectoderm, and Pitx2 in the right coelom, was as previously observed in other sea urchins. Conclusion: That genes associated with Nodal and BMP2/4 signalling are expressed in the hydrocoele lobes, indicates that they have a role in the developmental transition to pentamery, contributing to our understanding of how the most unusual body plan in the Bilateria may have evolved. We suggest that the Nodal and BMP2/4 signalling cascades might have been duplicated or split during the evolution to pentamery. [ABSTRACT FROM AUTHOR]

Summary Many animals are indirect developers with distinct larval and adult body plans [ 1 ]. The molecular basis of differences between larval and adult forms is often poorly understood, adding a level of uncertainty to comparative developmental studies that use data from both indirect and direct developers. Here we compare the larval and adult body plans of an indirect developing hemichordate, Schizocardium californicum [ 2 ]. We describe the expression of 27 transcription factors with conserved roles in deuterostome ectodermal anteroposterior (AP) patterning in developing embryos, tornaria larvae, and post-metamorphic juveniles and show that the tornaria larva of S. californicum is transcriptionally similar to a truncated version of the adult. The larval ectoderm has an anterior molecular signature, while most of the trunk, defined by the expression of hox1 – 7 , is absent. Posterior ectodermal activation of Hox is initiated in the late larva prior to metamorphosis, in preparation for the transition to the adult form, in which the AP axis converges on a molecular architecture similar to that of the direct developing hemichordate Saccoglossus kowalevskii . These results identify a molecular correlate of a major difference in body plan between hemichordate larval and adult forms and confirm the hypothesis that deuterostome larvae are “swimming heads” [ 3 ]. This will allow future comparative studies with hemichordates to take into account molecular differences caused by early life history evolution within the phylum. Additionally, comparisons with other phyla suggest that a delay in trunk development is a feature of indirect development shared across distantly related phyla. [ABSTRACT FROM AUTHOR]

Background: Head or anterior body part regeneration is commonly associated with protostome, but not deuterostome invertebrates. However, it has been shown that the solitary hemichordate Ptychodera flava possesses the remarkable capacity to regenerate their entire nervous system, including their dorsal neural tube and their anterior head-like structure, or proboscis. Hemichordates, also known as acorn worms, are marine invertebrate deuterostomes that have retained chordate traits that were likely present in the deuterostome ancestor, placing these animals in a vital position to study regeneration and chordate evolution. All acorn worms have a tripartite body plan, with an anterior proboscis, middle collar region, and a posterior trunk. The collar houses a hollow, dorsal neural tube in ptychoderid hemichordates and numerous chordate genes involved in brain and spinal cord development are expressed in a similar anterior-posterior spatial arrangement along the body axis. Results: We have examined anterior regeneration in the hemichordate Ptychodera flava and report the spatial and temporal morphological changes that occur. Additionally, we have sequenced, assembled, and analyzed the transcriptome for eight stages of regenerating P. flava, revealing significant differential gene expression between regenerating and control animals. Conclusions: Importantly, we have uncovered developmental steps that are regeneration-specific and do not strictly follow the embryonic program. Developmental Dynamics 245:1159-1175, 2016. © 2016 The Authors. Developmental Dynamics published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists [ABSTRACT FROM AUTHOR]

The mechanisms that underlie directional cell migration are incompletely understood. Eph receptors usually guide migrations of cells by exclusion from regions expressing Ephrin. In sea urchin embryos, pigmented immunocytes are specified in vegetal epithelium, transition to mesenchyme, migrate, and re-enter ectoderm, distributing in dorsal ectoderm and ciliary band, but not ventral ectoderm. Immunocytes express Sp-Eph and Sp-Efn is expressed throughout dorsal and ciliary band ectoderm. Interfering with expression or function of Sp-Eph results in rounded immunocytes entering ectoderm but not adopting a dendritic form. Expressing Sp-Efn throughout embryos permits immunocyte insertion in ventral ectoderm. In mosaic embryos, immunocytes insert preferentially in ectoderm expressing Sp-Efn. We conclude that Sp-Eph signaling is necessary and sufficient for epithelial insertion. As well, we propose that immunocytes disperse when Sp-Eph enhances adhesion, causing haptotactic movement to regions of higher ligand abundance. This is a distinctive example of Eph/Ephrin signaling acting positively to pattern migrating cells.

Neuropeptides are evolutionarily ancient mediators of neuronal signalling in nervous systems. With recent advances in genomics/transcriptomics, an increasingly wide range of species has become accessible for molecular analysis. The deuterostomian invertebrates are of particular interest in this regard because they occupy an ‘intermediate' position in animal phylogeny, bridging the gap between the well-studied model protostomian invertebrates (e.g. Drosophila melanogaster, Caenorhabditis elegans) and the vertebrates. Here we have identified 40 neuropeptide precursors in the starfish Asterias rubens, a deuterostomian invertebrate from the phylum Echinodermata. Importantly, these include kisspeptin-type and melanin-concentrating hormone-type precursors, which are the first to be discovered in a non-chordate species. Starfish tachykinin-type, somatostatin-type, pigment-dispersing factor-type and corticotropin-releasing hormone-type precursors are the first to be discovered in the echinoderm/ambulacrarian clade of the animal kingdom. Other precursors identified include vasopressin/oxytocin-type, gonadotropin-releasing hormone-type, thyrotropin-releasing hormone-type, calcitonin-type, cholecystokinin/gastrin-type, orexin-type, luqin-type, pedal peptide/orcokinin-type, glycoprotein hormone-type, bursicon-type, relaxin-type and insulin-like growth factor-type precursors. This is the most comprehensive identification of neuropeptide precursor proteins in an echinoderm to date, yielding new insights into the evolution of neuropeptide signalling systems. Furthermore, these data provide a basis for experimental analysis of neuropeptide function in the unique context of the decentralized, pentaradial echinoderm bauplan.

E-proteins are basic helix-loop-helix (bHLH) transcription factors with essential roles in animal development. In mammals, these are encoded by three loci: E2-2 ( ITF-2/ME2/SEF2/TCF4 ) , E2A ( TCF3 ) , and HEB ( ME1/REB/TCF12 ). The HEB and E2-2 paralogs are expressed as alternative (Alt) isoforms with distinct N-terminal sequences encoded by unique exons under separate regulatory control. Expression of these alternative transcripts is restricted relative to the longer (Can) forms, suggesting distinct regulatory roles, although the functions of the Alt proteins remain poorly understood. Here, we characterize the single sea urchin E-protein ortholog ( SpE-protein ). The organization of the SpE-protein gene closely resembles that of the extended HEB/E2-2 vertebrate loci, including a transcript that initiates at a homologous alternative transcription start site ( SpE-Alt ). The existence of an Alt form in the sea urchin indicates that this feature predates the emergence of the vertebrates. We present additional evidence indicating that this transcript was present in the common bilaterian ancestor. In contrast to the widely expressed canonical form ( SpE-Can ), SpE-Alt expression is tightly restricted. SpE-Alt is expressed in two phases: first in aboral non-skeletogenic mesenchyme (NSM) cells and then in oral NSM cells preceding their differentiation and ingression into the blastocoel. Derivatives of these cells mediate immune response in the larval stage. Inhibition of SpE-Alt activity interferes with these events. Notably, although the two isoforms are initially co-expressed, as these cells differentiate, SpE-Can is excluded from the SpE-Alt + cell population. This mutually exclusive expression is dependent on SpE-Alt function, which reveals a previously undescribed negative regulatory linkage between the two E-protein forms. Collectively, these findings reorient our understanding of the evolution of this transcription factor family and highlight fundamental properties of E-protein biology. [ABSTRACT FROM AUTHOR]

Metchnikoff’s use of sea star larvae to observe encapsulation and phagocytosis, which was followedmuch later by allograft rejection kinetics, revealed that echinoderms had an innate immune system thatwas lacking of adaptive attributes. Larval sea urchins mount defenses in response to contact withmicrobes, which are mediated by phagocytic blastocoelar cells and pigment cells. In the adult, thecoelomocytes mediate immune responses through phagocytosis and encapsulation of foreign particles inaddition to degranulation of antimicrobial molecules. Molecular analysis of immune functions in the seaurchin has demonstrated a complement system that appears to have multiple alternative pathways andseveral activators of the lectin pathway, but may be missing the terminal pathway. Other genes andproteins involved in the sea urchin immunity include expanded sets of lectins, proteins with scavengerreceptor cysteine-rich repeats, Toll-like receptors and associated signalling proteins. A vast array ofproteins belonging to the 185/333 family are expressed in coelomocytes in response to lipopolysaccharideand show a surprising level of diversity. The sea urchin innate immune system has a number of largegene families with unexpected complexities and elevated levels of diversification.

Background: Establishment and maintenance of cell polarity is critical for normal embryonic development. Previously, it was thought that the echinoderm embryo remained relatively unpolarized until the first asymmetric division at the 16-cell stage. Here, we analyzed roles of the cell polarity regulators, the PAR complex proteins, and how their disruption in early development affects later developmental milestones. Results: We found that PAR6, aPKC, and CDC42 localize to the apical cortex as early as the 2-cell stage and that this localization is retained through the gastrula stage. Of interest, PAR1 also colocalizes with these apical markers through the gastrula stage. Additionally, PAR1 was found to be in complex with aPKC, but not PAR6. PAR6, aPKC, and CDC42 are anchored in the cortical actin cytoskeleton by assembled myosin. Furthermore, assembled myosin was found to be necessary to maintain proper PAR6 localization through subsequent cleavage divisions. Interference with myosin assembly prevented the embryos from reaching the blastula stage, while transient disruptions of either actin or microtubules did not have this effect. Conclusions: These observations suggest that disruptions of the polarity in the early embryo can have a significant impact on the ability of the embryo to reach later critical stages in development. [ABSTRACT FROM AUTHOR]

Five families of neuropeptides that have a C-terminal RFamide motif have been identified in vertebrates: 1. gonadotropin-inhibitory hormone (GnIH), 2. neuropeptide FF (NPFF) 3. pyroglutamylated RFamide peptide (QRFP), 4. prolactin-releasing peptide (PrRP) and 5. Kisspeptin. Experimental demonstration of neuropeptide-receptor pairings combined with comprehensive analysis of genomic and/or transcriptomic sequence data indicate that, with the exception of the deuterostomian PrRP system, the evolutionary origins of these neuropeptides can be traced back to the common ancestor of bilaterians. Here we review the occurrence of homologs of vertebrate RFamide-type neuropeptides and their receptors in deuterostomian invertebrates - urochordates, cephalochordates, hemichordates and echinoderms. Extending analysis of the occurrence of the RFamide motif in other bilaterian neuropeptide families reveals RFamide-type peptides that have acquired modified C-terminal characteristics in the vertebrate lineage (e.g. NPY/NPF), neuropeptide families where the RFamide motif is unique to protostomian members (e.g. CCK/sulfakinins) and RFamide-type peptides that have been lost in the vertebrate lineage (e.g. luqins). Furthermore, the RFamide motif is also a feature of neuropeptide families with a more restricted phylogenetic distribution (e.g. the prototypical FMRFamide-related neuropeptides in protostomes). Thus, the RFamide motif is both an ancient and a convergent feature of neuropeptides, with conservation, acquisition or loss of this motif occurring in different branches of the animal kingdom.

Concerning the evolution of deuterostomes, enteropneusts (acorn worms) occupy a pivotal role as they share some characteristics with chordates (e.g., tunicates and vertebrates) but are also closely related to echinoderms (e.g., sea urchin). The nervous system in particular can be a highly informative organ system for evolutionary inferences, and advances in fluorescent microscopy have revealed overwhelming data sets on neurogenesis in various clades. However, immunocytochemical descriptions of neurogenesis of juvenile enteropneusts are particularly scarce, impeding the reconstruction of nervous system evolution in this group. We followed morphogenesis of the nervous system in two enteropneust species, one with direct ( Saccoglossus kowalevskii) and the other with indirect development ( Balanoglossus misakiensis), using an antibody against serotonin and electron microscopy. We found that all serotonin-like immunoreactive (LIR) neurons in both species are bipolar ciliary neurons that are intercalated between other epidermal cells. Unlike the tornaria larva of B. misakiensis, the embryonic nervous system of S. kowalevskii lacks serotonin-LIR neurons in the apical region as well as an opisthotroch neurite ring. Comparative analysis of both species shows that the projections of the serotonin-LIR somata initially form a basiepidermal plexus throughout the body that disappears within the trunk region soon after settlement before the concentrated dorsal and ventral neurite bundles emerge. Our data reveal a highly conserved mode of neurogenesis in enteropneusts that is independent of the developing mode and is inferred to be a common feature for Enteropneusta. Moreover, all detected serotonin-LIR neurons are presumably receptor cells, and the absence of serotonin-LIR interneurons from the enteropneust nervous system, which are otherwise common in various bilaterian central nervous systems, is interpreted as a loss that might have occurred already in the last common ancestor of Ambulacraria. [ABSTRACT FROM AUTHOR]

Enteropneusts or acorn worms are marine deuterostomes that have retained many plesiomorphic characters. Thus, enteropneusts are of prime interest in evolutionary comparisons between deuterostomes and protostomes. In the present study, the larval eyes of Glossobalanus marginatus were reconstructed and described based on serial sectioning for transmission electron microscopy. The everse eyes of the late Metschnikoff/early Krohn-stage tornaria larvae of G. marginatus are epidermal structures consisting of two rows of in total 13 shading pigment cells and another two rows of 13 photoreceptor cells. The pigment cells form a shallow cup with a relatively wide opening, making the cup-shaped eye optically unsuitable for picture generation. We demonstrate that the photosensitive cells possess numerous enlarged microvilli and an unmodified apical cilium. Our ultrastructural studies thus corroborate the photoreceptor cells in the eye of G. marginatus to be of a clearly rhabdomeric type. Preliminary immunohistochemical experiments support those findings by demonstrating immunopositive reaction of the tornarian eye photoreceptors with an antibody designed against rhabdomeric sea urchin photopigment (Sp-Opsin4). Observations of living animals indicate that Late Metschnikoff/early Krohn-stage tornaria larvae are negatively phototactic, probably concordant with imminent metamorphosis. [ABSTRACT FROM AUTHOR]

Invertebrate animals are characterized by extraordinary diversity in terms of body plan, life history and life span. The past impression that invertebrate immune responses are controlled by relatively simple innate systems is increasingly contradicted by genomic analyses that reveal significant evolutionary novelty and complexity. One accessible measure of this complexity is the multiplicity of genes encoding homologs of pattern recognition receptors. These multigene families vary significantly in size, and their sequence character suggests that they vary in function. At the same time, certain aspects of downstream signaling appear to be conserved. Here, we analyze five major classes of immune recognition receptors from newly available animal genome sequences. These include the Toll-like receptors (TLR), Nod-like receptors (NLR), SRCR domain scavenger receptors, peptidoglycan recognition proteins (PGRP), and Gram negative binding proteins (GNBP). We discuss innate immune complexity in the invertebrate deuterostomes, which was first recognized in sea urchins, within the wider context of emerging genomic information across animal phyla. [ABSTRACT FROM AUTHOR]

Debate over the origin and evolution of vertebrates has occupied biologists and palaeontologists alike for centuries. This debate has been refined by molecular phylogenetics, which has resolved the place of vertebrates among their invertebrate chordate relatives, and that of chordates among their deuterostome relatives. The origin of vertebrates is characterized by wide-ranging genomic, embryologic and phenotypic evolutionary change. Analyses based on living lineages suggest dramatic shifts in the tempo of evolutionary change at the origin of vertebrates and gnathostomes, coincident with whole-genome duplication events. However, the enriched perspective provided by the fossil record demonstrates that these apparent bursts of anatomical evolution and taxic richness are an artefact of the extinction of phylogenetic intermediates whose fossil remains evidence the gradual assembly of crown gnathostome characters in particular. A more refined understanding of the timing, tempo and mode of early vertebrate evolution rests with: (1) better genome assemblies for living cyclostomes; (2) a better understanding of the anatomical characteristics of key fossil groups, especially the anaspids, thelodonts, galeaspids and pituriaspids; (3) tests of the monophyly of traditional groups; and (4) the application of divergence time methods that integrate not just molecular data from living species, but also morphological data and extinct species. The resulting framework will provide for rigorous tests of rates of character evolution and diversification, and of hypotheses of long-term trends in ecological evolution that themselves suffer for lack of quantitative functional tests. The fossil record has been silent on the nature of the transition from jawless vertebrates to the jawed vertebrates that have dominated communities since the middle Palaeozoic. Elucidation of this most formative of episodes likely rests with the overhaul of early vertebrate systematics that we propose, but perhaps more fundamentally with fossil grades that await discovery. [ABSTRACT FROM AUTHOR]

Abstract: Background: Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes. Methods: PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC-MS. Results: Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26μM for ketoconazole and 0.64μM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51s. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s. Conclusions: Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s. General significance: The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications. [Copyright &y& Elsevier]

Five families of neuropeptides that have a C-terminal RFamide motif have been identified in vertebrates: (1) gonadotropin-inhibitory hormone (GnIH), (2) neuropeptide FF (NPFF), (3) pyroglutamylated RFamide peptide (QRFP), (4) prolactin-releasing peptide (PrRP), and (5) Kisspeptin. Experimental demonstration of neuropeptide-receptor pairings combined with comprehensive analysis of genomic and/or transcriptomic sequence data indicate that, with the exception of the deuterostomian PrRP system, the evolutionary origins of these neuropeptides can be traced back to the common ancestor of bilaterians. Here, we review the occurrence of homologs of vertebrate RFamide-type neuropeptides and their receptors in deuterostomian invertebrates - urochordates, cephalochordates, hemichordates, and echinoderms. Extending analysis of the occurrence of the RFamide motif in other bila-terian neuropeptide families reveals RFamide-type peptides that have acquired modified C-terminal characteristics in the vertebrate lineage (e.g., NPY/NPF), neuropeptide families where the RFamide motif is unique to protostomian members (e.g., CCK/sulfakinins), and RFamide-type peptides that have been lost in the vertebrate lineage (e.g., luqins). Furthermore, the RFamide motif is also a feature of neuropeptide families with a more restricted phylogenetic distribution (e.g., the prototypical FMRFamide-related neuropeptides in protostomes).Thus, the RFamide motif is both an ancient and a convergent feature of neuropeptides, with conservation, acquisition, or loss of this motif occurring in different branches of the animal kingdom. [ABSTRACT FROM AUTHOR]

Abstract: Defining the organization and temporal onset of key steps in neurogenesis in invertebrate deuterostomes is critical to understand the evolution of the bilaterian and deuterostome nervous systems. Although recent studies have revealed the organization of the nervous system in adult hemichordates, little attention has been paid to neurogenesis during embryonic development in this third major phylum of deuterostomes. We examine the early events of neural development in the enteropneust hemichordate Saccoglossus kowalevskii by analyzing the expression of 11 orthologs of key genes associated with neurogenesis in an expansive range of bilaterians. Using in situ hybridization (ISH) and RT-PCR, we follow the course of neural development to track the transition of the early embryonic diffuse nervous system to the more regionalized midline nervous system of the adult. We show that in Saccoglossus, neural progenitor markers are expressed maternally and broadly encircle the developing embryo. An increase in their expression and the onset of pan neural markers, indicate that neural specification occurs in late blastulae – early gastrulae. By mid-gastrulation, punctate expression of markers of differentiating neurons encircling the embryo indicate the presence of immature neurons, and at the end of gastrulation when the embryo begins to elongate, markers of mature neurons are expressed. At this stage, expression of a subset of neuronal markers is concentrated along the trunk ventral and dorsal midlines. These data indicate that the diffuse embryonic nervous system of Saccoglossus is transient and quickly reorganizes before hatching to resemble the adult regionalized, centralized nervous system. This regionalization occurs at a much earlier developmental stage than anticipated indicating that centralization is not linked in S. kowalevskii to a lifestyle change of a swimming larva metamorphosing to a crawling worm-like adult. [Copyright &y& Elsevier]

Abstract: The structure and development of the larval nervous systems of all classes of echinoderms have been described and details of embryonic signaling mechanisms patterning neurogenesis have been revealed experimentally in sea urchins. Several features of neuroanatomy and neural development indicate that echinoids are the most derived group. Here we describe the development and organization of the nervous system of a cidaroid, Eucidaris tribuloides. The cidaroids are one of two major clades of echinoids, and are considered to have features of anatomy and development that represent the common ancestor to all echinoids. The embryos of E. tribuloides lack a thickened animal plate and serotonergic neurons arise laterally, associated with the ciliary band. Although lacking a discrete apical organ, plutei have serotonergic neurons associated with the pre-oral ciliary band joined by a few diffusely arranged connecting axons. Chordin and Hnf6, early markers for oral ectoderm and ciliary band, are expressed in similar patterns to euechinoids. However, an animal pole domain marker, Nk2.1, is expressed in a broader region of anterior ectoderm than in euechinoids. Six3, a proneural marker that is restricted to the animal plate of euechinoids, is expressed laterally in the preoral ciliary band at the same location as the serotonergic neurons. We conclude that the organization and development of the larval nervous system of E. tribuloides retains features shared with other echinoderm larvae, but not with euechinoids. These data support a model in which several distinctive features of euechinoid neural organization are derived, having arisen after the divergence of the two clades of echinoids about 265 million years ago. We hypothesize that differences in the developmental mechanisms that restrict neurogenesis to the animal pole forms the basis for the distinctive neuroanatomy of euechinoids. [Copyright &y& Elsevier]

Respiratory trees of holothurians are blind-ended evaginations of the posterior digestive system that are rhythmically inflated with seawater via the anus, and are considered to have respiratory and excretory functions. We tested the assimilatory capability of the respiratory tree epithelium by exposing adults of the oral deposit-feeding aspidochirotid sea cucumber Parastichopus californicus to (1) 14C-labeled unicellular algae and (2) iron-containing macromolecules, and then following the distribution of the labels in various body regions. The abundance of the 14C (nmol 14C g wet weight−1) varied among body regions. The majority of the label was associated with the respiratory trees. The next greatest concentration was in the rete mirabile of the hemal system. The amount of 14C within samples of the anterior digestive system, posterior digestive system, and body wall muscle at each sampling time was lower than that of the respiratory trees. The presence and abundance of the 14C label in samples of the hemal system and the physical association between the respiratory tree and the rete mirabile suggest initial uptake by the respiratory tree and subsequent transfer to the hemal system. The iron label from the protein ferritin and the polysaccharide iron dextran entered cells of the respiratory tree luminal epithelium. The presence of the iron label in mesenchyme cells within the connective tissue compartment of the respiratory trees exposed to ferritin further suggests directional transfer of materials. Holothurians with respiratory trees are nutritionally bipolar; the anus serves as a second mouth. [ABSTRACT FROM AUTHOR]

FGFs act in vertebrate mesoderm induction and also play key roles in early mesoderm formation in ascidians and amphioxus. However, in sea urchins initial characterizations of FGF function do not support a role in early mesoderm induction, making the ancestral roles of FGF signaling and mechanisms of mesoderm specification in deuterostomes unclear. In order to better characterize the evolution of mesoderm formation, we have examined the role of FGF signaling during mesoderm development in Saccoglossus kowalevskii, an experimentally tractable representative of hemichordates. We report the expression of an FGF ligand, fgf8/17/18, in ectoderm overlying sites of mesoderm specification within the archenteron endomesoderm. Embryological experiments demonstrate that mesoderm induction in the archenteron requires contact with ectoderm, and loss-of-function experiments indicate that both FGF ligand and receptor are necessary for mesoderm specification, fgf8/17/18 gain-of-function experiments establish that FGF8/17/18 is sufficient to induce mesoderm in adjacent endomesoderm. These experiments suggest that FGF signaling is necessary from the earliest stages of mesoderm specification and is required for all mesoderm development. Furthermore, they suggest that the archenteron is competent to form mesoderm or endoderm, and that FGF signaling from the ectoderm defines the location and amount of mesoderm. When considered in a comparative context, these data support a phylogenetically broad requirement for FGF8/17/18 signaling in mesoderm specification and suggest that FGF signaling played an ancestral role in deuterostome mesoderm formation. [ABSTRACT FROM AUTHOR]

Early coelomic development in the abbreviated development of the sea urchin Holopneustes purpurescens is described and then used in a comparison with coelomic development in chordate embryos to support homology between a single arm of the five-armed radial body plan of an echinoderm and the single bilateral axis of a chordate. The homology depends on a positional similarity between the origin of the hydrocoele in echinoderm development and the origin of the notochord in chordate development, and a positional similarity between the respective origins of the coelomic mesoderm and chordate mesoderm in echinoderm and chordate development. The hydrocoele is homologous with the notochord and the secondary podia are homologous with the somites. The homology between a single echinoderm arm and the chordate axis becomes clear when the aboral to oral growth from the archenteron in the echinoderm larva is turned anteriorly, more in line with the anterior-posterior axis of the early zygote. A dorsoventral axis inversion in chordates is not required in the proposed homology. [ABSTRACT FROM AUTHOR]

Metchnikoff’s use of sea star larvae to observe encapsulation and phagocytosis, which was followed much later by allograft rejection kinetics, revealed that echinoderms had an innate immune system that was lacking of adaptive attributes. Larval sea urchins mount defenses in response to contact with microbes, which are mediated by phagocytic blastocoelar cells and pigment cells. In the adult, the coelomocytes mediate immune responses through phagocytosis and encapsulation of foreign particles in addition to degranulation of antimicrobial molecules. Molecular analysis of immune functions in the sea urchin has demonstrated a complement system that appears to have multiple alternative pathways and several activators of the lectin pathway, but may be missing the terminal pathway. Other genes and proteins involved in the sea urchin immunity include expanded sets of lectins, proteins with scavenger receptor cysteine-rich repeats, Toll-like receptors and associated signalling proteins. A vast array of proteins belonging to the 185/333 family are expressed in coelomocytes in response to lipopolysaccharide and show a surprising level of diversity. The sea urchin innate immune system has a number of large gene families with unexpected complexities and elevated levels of diversification.

Hemichordates are a deuterostome phylum, the sister group to echinoderms, and closely related to chordates. They have thus been used to gain insights into the origins of deuterostome and chordate body plans. Developmental studies of this group have a long and distinguished history. Recent improvements in animal husbandry, functional tool development and genomic resources have resulted in novel developmental data from several species in this group. In this Primer, we introduce representative hemichordate species with contrasting modes of development and summarize recent findings that are beginning to yield important insights into deuterostome developmental mechanisms. [ABSTRACT FROM AUTHOR]

Hemichordate worms possess ciliated gills on their trunk, and the homology of these structures with the pharyngeal gill slits of chordates has long been a topic of debate in the fields of evolutionary biology and comparative anatomy. Here, we show conservation of transcription factor expression between the developing pharyngeal gill pores of the hemichordate Saccoglossus kowalevskii and the pharyngeal gill slit precursors (i.e. pharyngeal endodermal outpockets) of vertebrates. Transcription factors that are expressed in the pharyngeal endoderm, ectoderm and mesenchyme of vertebrates are expressed exclusively in the pharyngeal endoderm of S. kowalevskii. The pharyngeal arches and tongue bars of S. kowalevskii lack Tbx1-expressing mesoderm, and are supported solely by an acellular collagenous endoskeleton and by compartments of the trunk coelom. Our findings suggest that hemichordate and vertebrate gills are homologous as simple endodermal outpockets from the foregut, and that much vertebrate pharyngeal complexity arose coincident with the incorporation of cranial paraxial mesoderm and neural crest-derived mesenchyme within pharyngeal arches along the chordate and vertebrate stems, respectively. [ABSTRACT FROM AUTHOR]

Stach, T. and Kaul, S. 2011. The postanal tail of the enteropneust Saccoglossus kowalevskii is a ciliary creeping organ without distinct similarities to the chordate tail. - Acta Zoologica (Stockholm) : 150-160. The postanal tail of chordates is one of the key characters in chordate evolution and it has been suggested to be homologous to the postanal tail of harrimaniid enteropneusts. We present electron microscopic data of the ontogeny of the postanal tail in the enteropneust Saccoglossus kowalevskii. The postanal tail develops as a ventral posterior allometric outgrowth with a ventral extension of the telotroch. Transmission electron microscopy of serial sections reveals the epidermal organization of the postanal tail with the exception of short, bilaterally symmetric extensions of the paired metacoels. The epidermis cells are connected by apical junctions, rest basally on the extracellular matrix surrounding the mesoderm, and possess a basiepidermal nerve net. The ventral cells in the postanal tail are multiciliated and used for creeping. Dorsal cells are monociliated with numerous microvilli. Two types of glandular cells are present among the epidermis cells. The mesoderm cells contain myofilaments. We were unable to detect anatomical structures similar to the ones present in the postanal locomotory tail of chordates, such as notochord, neural tube, or endodermal strand. Thus, results of our anatomical study do not support homology of the postanal chordate tail and the postanal tail of harrimaniid enteropneusts. [ABSTRACT FROM AUTHOR]

The availability of whole genome sequences from multiple metazoan phyla is making it possible to determine their phylogeny. We have found that a sea urchin and human define a clade that excludes a tunicate, contradicting both classical and recent molecular studies that place the tunicate and vertebrate in the Chordate phylum. Intriguingly, by means of a novel four taxa analysis, we have partitioned the 2000 proteins responsible for this assignment into two groups. One group, containing about 40% of the proteins, supports the classical assemblage of the tunicate with vertebrates, while the remaining group places the tunicate outside of the chordate assemblage. The existence of these two phylogenetic groups is robustly maintained in five, six and nine taxa analyses. These results suggest that major horizontal gene transfer events occurred during the emergence of one of the metazoan phyla. The simplest explanation is that the modern tunicate (as represented by Ciona intestinalis) began as a hybrid between a primitive vertebrate and some other organism, perhaps from an extinct and unidentified protostome phylum, at a time close to but after the diversification of the chordates and echinoderms and before the lineages leading to Drosophila melanogaster and Caenorhabditis elegans diverged. [ABSTRACT FROM AUTHOR]

Abstract: In protostomes, cell polarity is present after fertilization whereas most deuterostome embryos show minimal polarity during the early cleavages. We now show establishment of cell polarity as early as the first cleavage division in sea urchin embryos. We find, using the apical markers GM1, integrins, and the aPKC–PAR6 complex, that cells are polarized upon insertion of distinct basolateral membrane at the first division. This early apical–basolateral polarity, similar to that found in much larger cleaving amphibian zygotes, reflects precocious functional epithelial cell polarity. Isolated cleavage blastomeres exhibit polarized actin-dependent fluid phase endocytosis only on the GM1, integrin, microvillus-containing apical surface. A role for a functional PAR complex in cleavage plane determination was shown with experiments interfering with aPKC activity, which results in several spindle defects and compromised blastula development. These studies suggest that cell and embryonic polarity is established at the first cleavage, mediated in part by the Par complex of proteins, and is achieved by directed insertion of basolateral membrane in the cleavage furrow. [Copyright &y& Elsevier]

The evolutionary origin of vertebrates has been debated ad nauseam by anatomists, paleontologists, embryologists, and physiologists, but it is only now that molecular phylogenetics is providing a more rigorous framework for the placement of vertebrates among their invertebrate relatives that we can begin to arrive at concrete conclusions concerning the nature of ancient ancestors and the sequence in which characteristic anatomical features were acquired. Vertebrates tunicates and cephalochordates together comprise the chordate phylum, which along with echinoderms and hemichordates constitute the deuterostomes. The origin of vertebrates and of jawed vertebrates is characterized by a doubling of the vertebrate genome, leading to hypotheses that this genomic event drove organismal macroevolution. However, this perspective of evolutionary history, based on living organisms alone, is an artifact. Phylogenetic trees that integrate fossil vertebrates among their living relatives demonstrate the gradual and piecemeal assembly of the gnathostome body plan. Unfortunately, it has not been possible to demonstrate gradual assembly of the vertebrate body plan. This is not because vertebrates are irreducibly complex but because many of the characters that distinguish vertebrates from invertebrates are embryological and cellular and, therefore, inherently unfossilizable. [ABSTRACT FROM AUTHOR]

Abstract: A gene encoding an ortholog of vertebrate CB1/CB2 cannabinoid receptors was recently identified in the urochordate Ciona intestinalis (CiCBR; [Elphick, M.R., Satou, Y., Satoh, N., 2003. The invertebrate ancestry of endocannabinoid signalling: an orthologue of vertebrate cannabinoid receptors in the urochordate Ciona intestinalis. Gene 302, 95–101.]). Here a cannabinoid receptor ortholog (BfCBR) has been identified in the cephalochordate Branchiostoma floridae. BfCBR is encoded by a single exon and is 410 amino acid residue protein that shares 28% sequence identity with CiCBR and 23% sequence identity with human CB1 and human CB2. The discovery of BfCBR and CiCBR and the absence of cannabinoid receptor orthologs in non-chordate invertebrates indicate that CB1/CB2-like cannabinoid receptors originated in an invertebrate chordate ancestor of urochordates, cephalochordates and vertebrates. Furthermore, analysis of the relationship of BfCBR and CiCBR with vertebrate CB1 and CB2 receptors indicates that the gene/genome duplication that gave rise to CB1 and CB2 receptors occurred in the vertebrate lineage. Identification of BfCBR, in addition to CiCBR, paves the way for comparative analysis of the expression and functions of these proteins in Branchiostoma and Ciona, respectively, providing an insight into the ancestral functions of cannabinoid receptors in invertebrate chordates prior to the emergence of CB1 and CB2 receptors in vertebrates. [Copyright &y& Elsevier]

Selective Na+-dependent re-uptake of biogenic monoamines at mammalian nerve synapses is accomplished by three types of solute-linked carrier family 6 (SLC6) membrane transporter with high affinity for serotonin (SERTs), dopamine (DATs) and norepinephrine (NETs). An additional SLC6 monoamine transporter (OAT), is responsible for the selective uptake of the phenolamines octopamine and tyramine by insect neurons. We have characterized a similar high-affinity phenoloamine transporter expressed in the CNS of the earthworm Lumbricus terrestris. Phylogenetic analysis of its protein sequence clusters it with both arthropod phenolamine and chordate catecholamine transporters. To clarify the relationships among metazoan monoamine transporters we identified representatives in the major branches of metazoan evolution by polymerase chain reaction (PCR)-amplifying conserved cDNA fragments from isolated nervous tissue and by analyzing available genomic data. Analysis of conserved motifs in the sequence data suggest that the presumed common ancestor of modern-day Bilateria expressed at least three functionally distinct monoamine transporters in its nervous system: a SERT currently found throughout bilaterian phyla, a DAT now restricted in distribution to protostome invertebrates and echinoderms and a third monoamine transporter (MAT), widely represented in contemporary Bilateria, that is selective for catecholamines and/or phenolamines. Chordate DATs, NETs, epinephrine transporters (ETs) and arthropod and annelid OATs all belong to the MAT clade. Contemporary invertebrate and chordate DATs belong to different SLC6 clades. Furthermore, the genes for dopamine and norepinephrine transporters of vertebrates are paralogous, apparently having arisen through duplication of an invertebrate MAT gene after the loss of an invertebrate-type DAT gene in a basal protochordate. [ABSTRACT FROM AUTHOR]

Abstract: The predicted gene models derived from the sea urchin genome were compared to the gene catalogs derived from other completed genomes. The models were categorized by their best match to conserved protein domains. Identification of potential orthologs and assignment of sea urchin gene models to groups of homologous genes was accomplished by BLAST alignment and through the use of a clustering algorithm. For the first time, an overview of the sea urchin genetic toolkit emerges and by extension a more precise view of the features shared among the gene catalogs that characterize the super-clades of animals: metazoans, bilaterians, chordate and non-chordate deuterostomes, ecdysozoan and lophotrochozoan protostomes. About one third of the 40 most prevalent domains in the sea urchin gene models are not as abundant in the other genomes and thus constitute expansions that are specific at least to sea urchins if not to all echinoderms. A number of homologous groups of genes previously restricted to vertebrates have sea urchin representatives thus expanding the deuterostome complement. Obversely, the absence of representatives in the sea urchin confirms a number of chordate specific inventions. The specific complement of genes in the sea urchin genome results largely from minor expansions and contractions of existing families already found in the common metazoan “toolkit” of genes. However, several striking expansions shed light on how the sea urchin lives and develops. [Copyright &y& Elsevier]

Abstract: The sequencing of the Strongylocentrotus purpuratus genome provides a unique opportunity to investigate the function and evolution of neural genes. The neurobiology of sea urchins is of particular interest because they have a close phylogenetic relationship with chordates, yet a distinctive pentaradiate body plan and unusual neural organization. Orthologues of transcription factors that regulate neurogenesis in other animals have been identified and several are expressed in neurogenic domains before gastrulation indicating that they may operate near the top of a conserved neural gene regulatory network. A family of genes encoding voltage-gated ion channels is present but, surprisingly, genes encoding gap junction proteins (connexins and pannexins) appear to be absent. Genes required for synapse formation and function have been identified and genes for synthesis and transport of neurotransmitters are present. There is a large family of G-protein-coupled receptors, including 874 rhodopsin-type receptors, 28 metabotropic glutamate-like receptors and a remarkably expanded group of 161 secretin receptor-like proteins. Absence of cannabinoid, lysophospholipid and melanocortin receptors indicates that this group may be unique to chordates. There are at least 37 putative G-protein-coupled peptide receptors and precursors for several neuropeptides and peptide hormones have been identified, including SALMFamides, NGFFFamide, a vasotocin-like peptide, glycoprotein hormones and insulin/insulin-like growth factors. Identification of a neurotrophin-like gene and Trk receptor in sea urchin indicates that this neural signaling system is not unique to chordates. Several hundred chemoreceptor genes have been predicted using several approaches, a number similar to that for other animals. Intriguingly, genes encoding homologues of rhodopsin, Pax6 and several other key mammalian retinal transcription factors are expressed in tube feet, suggesting tube feet function as photosensory organs. Analysis of the sea urchin genome presents a unique perspective on the evolutionary history of deuterostome nervous systems and reveals new approaches to investigate the development and neurobiology of sea urchins. [Copyright &y& Elsevier]

Abstract: Two distinct modes of germ line determination are used throughout the animal kingdom: conditional—an inductive mechanism, and autonomous—an inheritance of maternal factors in early development. This study identifies homologs of germ line determinants in the sea urchin Strongylocentrotus purpuratus to examine its mechanism of germ line determination. A list of conserved germ-line associated genes from diverse organisms was assembled to search the S. purpuratus genome for homologs, and the expression patterns of these genes were examined during embryogenesis by whole mount in situ RNA hybridization and QPCR. Of the 14 genes tested, all transcripts accumulate uniformly during oogenesis and Sp-pumilio, Sp-tudor, Sp-MSY, and Sp-CPEB1 transcripts are also uniformly distributed during embryonic development. Sp-nanos2, Sp-seawi, and Sp-ovo transcripts, however, are enriched in the vegetal plate of the mesenchyme blastula stage and Sp-vasa, Sp-nanos2, Sp-seawi, and Sp-SoxE transcripts are localized in small micromere descendents at the tip of the archenteron during gastrulation and are then enriched in the left coelomic pouch of larvae. The results of this screen suggest that sea urchins conditionally specify their germ line, and support the hypothesis that this mechanism is the basal mode of germ line determination amongst deuterostomes. Furthermore, accumulation of germ line determinants selectively in small micromere descendents supports the hypothesis that these cells contribute to the germ line. [Copyright &y& Elsevier]

Abstract: The Receptor Tyrosine kinase (RTK) and TGF-β signaling pathways play essential roles during development in many organisms and regulate a plethora of cellular responses. From the genome sequence of Strongylocentrotus purpuratus, we have made an inventory of the genes encoding receptor tyrosine kinases and their ligands, and of the genes encoding cytokines of the TGF-β superfamily and their downstream components. The sea urchin genome contains at least 20 genes coding for canonical receptor tyrosine kinases. Seventeen of the nineteen vertebrate RTK families are represented in the sea urchin. Fourteen of these RTK among which ALK, CCK4/PTK7, DDR, EGFR, EPH, LMR, MET/RON, MUSK, RET, ROR, ROS, RYK, TIE and TRK are present as single copy genes while pairs of related genes are present for VEGFR, FGFR and INSR. Similarly, nearly all the subfamilies of TGF-β ligands identified in vertebrates are present in the sea urchin genome including the BMP, ADMP, GDF, Activin, Myostatin, Nodal and Lefty, as well as the TGF-β sensu stricto that had not been characterized in invertebrates so far. Expression analysis indicates that the early expression of nodal, BMP2/4 and lefty is restricted to the oral ectoderm reflecting their role in providing positional information along the oral–aboral axis of the embryo. The coincidence between the emergence of TGF-β-related factors such as Nodal and Lefty and the emergence of the deuterostome lineage strongly suggests that the ancestral function of Nodal could have been related to the secondary opening of the mouth which characterizes this clade, a hypothesis supported by functional data in the extant species. The sea urchin genome contains 6 genes encoding TGF-β receptors and 4 genes encoding prototypical Smad proteins. Furthermore, most of the transcriptional activators and repressors shown to interact with Smads in vertebrates have orthologues in echinoderms. Finally, the sea urchin genome contains an almost complete repertoire of genes encoding extracellular modulators of BMP signaling including Chordin, Noggin, Sclerotin, SFRP, Gremlin, DAN and Twisted gastrulation. Taken together, these findings indicate that the sea urchin complement of genes of the RTK and TGF-β signaling pathways is qualitatively very similar to the repertoire present in vertebrates, and that these genes are part of the common genetool kit for intercellular signaling of deuterostomes. [Copyright &y& Elsevier]

Abstract: Neural patterning genes that are expressed along the anterior–posterior axis of deuterostomes are expressed late in larval development in echinoderms and are thought to function in establishing the highly-derived, adult body plan. We have used genomic resources to clone an engrailed gene (SpEn) from Strongylocentrotus purpuratus, and with this we have developed an antibody specific for SpEn. SpEn is expressed late in embryogenesis in the developing larval nervous system. At the prism stage, a small number of neuroblasts in the oral ectoderm on the edge of the larval mouth begin expressing SpEn. The cells are in bilaterally symmetric positions. The expression of SpEn precedes the expression of the neural markers, synaptotagmin and serotonin in the SpEn immunoreactive cells. The SpEn cells are located on the margin of the domain of cells expressing SpNK2.1, but they do not have nuclear SpNK2.1. Expression of engrailed in a pair of bilateral neural structures in early development appears to be a shared feature of bilaterians. [Copyright &y& Elsevier]

Abstract: The starfish Asterias rubens is an invertebrate deuterostome whose nervous system shows remarkable regenerative properties. To understand when full functionality of a damaged part of the nervous system recovers, and to follow nerve regeneration in detail, we carried out behavioural experiments with 29 starfishes that had the nerve in one of the arms sectioned in a mid-arm position. Loss and recovery of normal behaviour was followed by video analysis of animal performance in an appetitive behavioural test. When compared to 13 control (unoperated) animals, the appetitive response of freshly sectioned animals is normal initially, progressively deteriorates up to 40 days after the lesion, and then gradually improves until 60 days, when recovery is complete. This is true only when one of the leading arms in the appetitive test is a sectioned arm; turning the starfish so that both the leading arms facing the prey are unlesioned, results in normal behaviour even at 40 days after the cut. Thus, regeneration is a multi-step process whose time course coincides with anatomical regeneration. At intermediate times the animals have coordination problems in an appetitive behaviour test and these give some insights into how arms may inter-communicate to organize concerted movements. [Copyright &y& Elsevier]

A new metazoan, Skeemella clavula gen. et sp. nov., is described from the Middle Cambrian Pierson Cove Formation of the Drum Mountains, Utah, USA. Skeemella is similar to vetulicolians, but differs from other examples of this group in the relative proportions of the anterior and posterior sections, the large number of divisions, and the elongate bifid termination. The posterior section is arthropodan in character. The similarity of this fossil to vetulicolians throws hypotheses of their deuterostome affinity into question and highlights their problematic status. [ABSTRACT FROM AUTHOR]

Abstract: Hedgehog (hh) is a multifunctional extracellular protein, and known as an essential signal molecule in morphogenetic movement in animal embryos. We have cloned, sequenced, and studied dynamic localization of Hphh, a hedgehog homologue of the sea urchin, Hemicentrotus pulcherrimus. The origin of Hphh transcribing cells was also verified during early embryogenesis. The amino acid sequence of Hphh shows high homology to Lvhh, an hh homologue cloned in the sea urchin, Lytechinus variegatus. Reverse transcriptase polymerase chain reaction showed that the transcription of Hphh occurred at and after 19h post-fertilization (19hpf) mesenchyme blastula stage until, at least, 69hpf 4-arm pluteus stage. Whole mount in situ hybridization showed Hphh transcription sites in a few cells at the tip of archenteron in 30hpf gastrulae. At around 45hpf 2-arm pluteus stage, the number of Hphh transcribed cells was 8, and unequally split to two groups, 5 cells in left coelomic sac and 3 cells in right coelomic sac. A cell lineage tracing by staining the small micromeres with 5-Bromo-2-deoxyuridine showed that Hphh was transcribed exclusively in all the small micromere descendants and comprised the coelomic sacs in 69hpf plutei. [Copyright &y& Elsevier]

Abstract: Retinoids in the organs (gonad [GND], body wall muscle [BWM], hepatopancreas [HP], gill, hemolymph cells and hemolymph plasma) of the adult ascidian Halocynthia roretzi were analyzed by high performance liquid chromatography. Retinal (RAL) occurred in every organ examined, and most of RAL (≥99%) was localized in the GND and BWM. None of the organs contained significant amounts of retinol (ROL) or retinyl ester (RE). Lipid droplets, which are characteristic of stellate cells (RE-storing cells of vertebrates), could not be found in the GND, BWM and HP by microscopic observations. These results indicate that this ascidian lacks the RE-storing mechanism, which is ubiquitous in adult vertebrates. The amount and localization of RAL showed the annual change in relation to the reproductive cycle. During summer, the growing season, RAL was present in both GND and BWM at a ratio of about 3:2. From summer to winter, RAL in the GND gradually increased, concomitant with the decrease of RAL in the BWM. In winter, the spawning season, most of RAL was present in the GND (ca. 98%). RAL appears to be accumulated first in the BWM and transported to oocytes accompanying yolk accumulation. ROL and RE were not implicated in the storage and transport of retinoids. The results in the present research strongly suggest that retinoic acid (RA) is produced by the two-step enzymatic reaction: carotenoid cleavage to RAL followed by RAL oxidation to RA and that the prevertebrate chordate lacks ROL-metabolizing systems. [Copyright &y& Elsevier]