Expression of the neuropeptide SALMFamide-1 during regeneration of the seastar radial nerve cord following arm autotomy.

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.

Acidified seawater impacts sea urchin larvae pH regulatory systems relevant for calcification.

Calcifying echinoid larvae respond to changes in seawater carbonate chemistry with reduced growth and developmental delay. To date, no information exists on how ocean acidification acts on pH homeostasis in echinoderm larvae. Understanding acid-base regulatory capacities is important because intracellular formation and maintenance of the calcium carbonate skeleton is dependent on pH homeostasis. Using H(+)-selective microelectrodes and the pH-sensitive fluorescent dye BCECF, we conducted in vivo measurements of extracellular and intracellular pH (pH(e) and pH(i)) in echinoderm larvae. We exposed pluteus larvae to a range of seawater CO(2) conditions and demonstrated that the extracellular compartment surrounding the calcifying primary mesenchyme cells (PMCs) conforms to the surrounding seawater with respect to pH during exposure to elevated seawater pCO(2). Using FITC dextran conjugates, we demonstrate that sea urchin larvae have a leaky integument. PMCs and spicules are therefore directly exposed to strong changes in pH(e) whenever seawater pH changes. However, measurements of pH(i) demonstrated that PMCs are able to fully compensate an induced intracellular acidosis. This was highly dependent on Na(+) and HCO(3)(-), suggesting a bicarbonate buffer mechanism involving secondary active Na(+)-dependent membrane transport proteins. We suggest that, under ocean acidification, maintained pH(i) enables calcification to proceed despite decreased pH(e). However, this probably causes enhanced costs. Increased costs for calcification or cellular homeostasis can be one of the main factors leading to modifications in energy partitioning, which then impacts growth and, ultimately, results in increased mortality of echinoid larvae during the pelagic life stage.

Brittlestars contain highly sulfated chondroitin sulfates/dermatan sulfates that promote fibroblast growth factor 2-induced cell signaling.

Glycosaminoglycans (GAGs) isolated from brittlestars, Echinodermata class Ophiuroidea, were characterized, as part of attempts to understand the evolutionary development of these polysaccharides. A population of chondroitin sulfate/dermatan sulfate (CS/DS) chains with a high overall degree of sulfation and hexuronate epimerization was the major GAG found, whereas heparan sulfate (HS) was below detection level. Enzymatic digestion with different chondroitin lyases revealed exceptionally high proportions of di- and trisulfated CS/DS disaccharides. The latter unit appears much more abundant in one of four individual species of brittlestars, Amphiura filiformis, than reported earlier in other marine invertebrates. The brittlestar CS/DS was further shown to bind to growth factors such as fibroblast growth factor 2 and to promote FGF-stimulated cell signaling in GAG-deficient cell lines in a manner similar to that of heparin. These findings point to a potential biological role for the highly sulfated invertebrate GAGs, similar to those ascribed to HS in vertebrates.

Homeobox genes expressed during echinoderm arm regeneration.

Regeneration in echinoderms has proved to be more amenable to study in the laboratory than the more classical vertebrate models, since the smaller genome size and the absence of multiple orthologs for different genes in echinoderms simplify the analysis of gene function during regeneration. In order to understand the role of homeobox-containing genes during arm regeneration in echinoderms, we isolated the complement of genes belonging to the Hox class that are expressed during this process in two major echinoderm groups: asteroids (Echinaster sepositus and Asterias rubens) and ophiuroids (Amphiura filiformis), both of which show an extraordinary capacity for regeneration. By exploiting the sequence conservation of the homeobox, putative orthologs of several Hox genes belonging to the anterior, medial, and posterior groups were isolated. We also report the isolation of a few Hox-like genes expressed in the same systems.

Induced spawning with gamete release from body ruptures during reproduction of Xenoturbella bocki.

Xenoturbella is a marine invertebrate with a simple body plan, with recent phylogenomic studies suggesting that it forms the phylum Xenacoelomorpha together with the acoelomorphs. The phylogenetic position of the phylum is still under debate, whether it is an early branching bilaterian or a sister group to the Ambulacraria. Phylogenetic traits often appear during development, and larva resembling the cnidarian planula has been reported for Xenoturbella. However, subsequent developmental studies on Xenoturbella have been scarce. This is mainly due to the difficulties in collecting and keeping adult animals, resulting in the lack of data on the reproduction of the animal, such as the breeding season and the spawning pattern. Here we report on the reproduction of X. bocki and confirm that its breeding season is winter. Spawning induction resulted in gametes being released from body ruptures and not the mouth. No evidence supported the animal as a simultaneous hermaphrodite.

Ten years of marine evolutionary biology-Challenges and achievements of a multidisciplinary research initiative.

The Centre for Marine Evolutionary Biology (CeMEB) at the University of Gothenburg, Sweden, was established in 2008 through a 10-year research grant of 8.7 m€ to a team of senior researchers. Today, CeMEB members have contributed >500 scientific publications, 30 PhD theses and have organised 75 meetings and courses, including 18 three-day meetings and four conferences. What are the footprints of CeMEB, and how will the centre continue to play a national and international role as an important node of marine evolutionary research? In this perspective article, we first look back over the 10 years of CeMEB activities and briefly survey some of the many achievements of CeMEB. We furthermore compare the initial goals, as formulated in the grant application, with what has been achieved, and discuss challenges and milestones along the way. Finally, we bring forward some general lessons that can be learnt from a research funding of this type, and we also look ahead, discussing how CeMEB's achievements and lessons can be used as a springboard to the future of marine evolutionary biology.

Deuterostome phylogeny reveals monophyletic chordates and the new phylum Xenoturbellida.

Deuterostomes comprise vertebrates, the related invertebrate chordates (tunicates and cephalochordates) and three other invertebrate taxa: hemichordates, echinoderms and Xenoturbella. The relationships between invertebrate and vertebrate deuterostomes are clearly important for understanding our own distant origins. Recent phylogenetic studies of chordate classes and a sea urchin have indicated that urochordates might be the closest invertebrate sister group of vertebrates, rather than cephalochordates, as traditionally believed. More remarkable is the suggestion that cephalochordates are closer to echinoderms than to vertebrates and urochordates, meaning that chordates are paraphyletic. To study the relationships among all deuterostome groups, we have assembled an alignment of more than 35,000 homologous amino acids, including new data from a hemichordate, starfish and Xenoturbella. We have also sequenced the mitochondrial genome of Xenoturbella. We support the clades Olfactores (urochordates and vertebrates) and Ambulacraria (hemichordates and echinoderms). Analyses using our new data, however, do not support a cephalochordate and echinoderm grouping and we conclude that chordates are monophyletic. Finally, nuclear and mitochondrial data place Xenoturbella as the sister group of the two ambulacrarian phyla. As such, Xenoturbella is shown to be an independent phylum, Xenoturbellida, bringing the number of living deuterostome phyla to four.

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L).

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens', was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.

Neural development of the brittlestar Amphiura filiformis.

Comparative features of the development of the larval nervous system of ophiuroids have the potential for resolving aspects of echinoderm evolution. In Amphiura filiformis serotonergic neural progenitors appear in the animal plate of late gastrulae. The serotonergic progenitors increase in number and become displaced to the aboral ectoderm side of the developing ciliary band. The ciliary band neurons appear as irregularly spaced neural progenitors on the oral side of the ciliary band lateral to the mouth. These cells extend neurites along the axis of the ciliary band, which meet at the center of the ventral transverse ciliary band. The larval nervous system begins as a U-shaped tract of axons that surrounds the oral field and tracts of axons and neurons in the ciliary bands of the larval arms are added. In addition, the larval nervous system has an extensive pre-oral neuropil, rings of nerves surrounding the anus and pyloric sphincters, and a plexus of axons that surround the esophagus. The nervous system of the juvenile develops beneath the oral ectoderm. The components of the adult nervous system: five segments of radial nerve, commissures that form the nerve ring, and podial nerves all appear as the juvenile develops. The larval nervous system begins to fragment and degenerate as the juvenile grows. The complete description of neural development of an ophiuroid reveals that the four classes so far investigated are consistent with phylogenies based on adult features and comparisons of neural organization help rationalize conflicting hypotheses of the evolution of larval forms in echinoderms.

Coelomocyte numbers and expression of HSP70 in wounded sea stars during hypoxia.

Hypoxia, mainly caused by eutrophication, is a common and growing problem on marine soft bottoms. Echinoderms are known for their ability to regenerate tissue after wounding but hypoxia has a negative influence on regeneration and also on reproduction in echinoderms. We have investigated the cellular and molecular responses to wounding stress and hypoxia in the sea star Asterias rubens by using the total coelomocyte count (TCC) and the expression of heat shock proteins (HSPs). As early as 1 h after wounding, sea stars under hypoxic conditions show significantly increased TCC and, after 6 h, cell numbers increase approximately two-fold. After a 3-h hypoxia exposure of wounded animals, Western blot analysis reveals highly elevated coelomocyte cytoplasmic HSP70 expression. Non-wounded sea stars exposed to hypoxia and wounded animals kept in normoxia show enhanced HSP70 expression only after 24 h. Immunocytochemical analysis has not demonstrated any translocation of HSP70 from the cytoplasm to the nucleus. We conclude that both wounding and hypoxia elicit a stress response in sea stars and that the combined stress produces synergistic effects that may inhibit the initial processes of wound healing and regeneration.

Coelomocytes and post-traumatic response in the common sea star Asterias rubens.

Coelomocytes are recognized as the main cellular component of the echinoderm immune system. They are the first line of defense and their number and type can vary dramatically during infections or following injury. Sea stars have been used as a model system to study the regeneration process after autotomy or predation. In the present study we examined the cellular and biochemical responses of coelomocytes from the European sea star Asterias rubens to traumatic stress using immunochemical and biochemical approaches. In terms of trauma and post-traumatic stress period, here we consider the experimental arm amputation and the repair phase involved in the first 24 hours post-amputation, which mimicked a natural predation event. Four cell morphotypes were distinguishable in the coelomic fluid of both control and post-traumatic-stressed animals (phagocytes, amoebocytes, vibratile cells, hemocytes), but phagocytes were the major components, accounting for about 95% of the total population. Thus, the effects measured relate to the overall population of coelomocytes. A modest increase in the total number of freely circulating coelomocytes was observed 6 hours post-amputation. Interestingly, a monoclonal antibody (McAb) to a sea urchin embryo adhesion protein (toposome) cross-reacted with isolated sea star coelomocytes and stained the coelomic epithelium of control animals with an increase in trauma-stressed arms. In addition, coelomocytes from trauma-stressed animals showed a time-dependent increase in Hsp70 levels, as detected by both immunocytochemistry and immunoblotting within 24 hours after arm tip amputation, with a peak at 6 hours after amputation. Our findings indicate a clear role for coelomocytes and classic stress molecules in the post-traumatic stress associated with the early repair phase of regeneration.

Alteration and recovery of appetitive behaviour following nerve section in the starfish Asterias rubens.

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.

Inhibitory effect of a SALMFamide neuropeptide on secretion of gonad-stimulating substance from radial nerves in the starfish Asterina pectinifera.

In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. The SALMFamide family is also known to an echinoderm neuropeptide. The present study examined effect of SALMFamide 1 (S1) on oocyte maturation of starfish Asterina pectinifera. Unlike GSS, S1 did not induce spawning in starfish ovary. In contrast, S1 was found to inhibit GSS secretion from radial nerves by treatment with high K+ concentration. Fifty percent inhibition was obtained by 0.1 mM S1. S1 did not have any effect on GSS- and 1-MeAde-induced oocyte maturation. Following incubation with a S1 antibody and subsequently with rhodamine-conjugated second antibody, neural networks were observed in ovaries. The networks were restricted mainly to their surface with little evidence of immunoreactivity inside the basement membranes. This indicates that neural networks are distributed in the ovarian wall. The result further suggests that S1 plays a role in oocyte maturation to regulate GSS secretion from the nervous system.

Evidence for dynamic and multiple roles for huntingtin in Ciona intestinalis.

Although mutations in the huntingtin gene (HTT) due to poly-Q expansion cause neuropathology in humans (Huntington's disease; HD), the normal function(s) of the gene and its protein (HTT) remain obscure. With new information from recently sequenced invertebrate genomes, the study of new animal models opens the possibility of a better understanding of HTT function and its evolution. To these ends, we studied huntingtin expression pattern and dynamics in the invertebrate chordate Ciona intestinalis. Ciona huntingtin (Ci-HTT) shows a biphasic expression pattern during larval development and prior to metamorphosis. A single form of huntingtin protein is present until the early larval stages, at which time two different mass proteins become evident in the metamorphically competent larva. An antibody against Ci-HTT labeled 50 cells in the trunk mesenchyme regions in pre-hatching and hatched larvae and probably represents the distribution of the light form of the protein. Dual labeling with anti-Ci-HTT and anti-aldoketoreductase confirmed the presence of Ci-HTT in mesenchyme cells. Suppression of Ci-HTT RNA by a morpholino oligonucleotide reduced the number and apparent mobility of Ci-HTT positive cells. In Ciona, HTT expression has a dynamic temporal and spatial expression pattern that in ontogeny precedes metamorphosis. Although our results may reflect a derived function for the protein in pre- and post-metamorphic events in Ciona, we also note that as in vertebrates, there is evidence for multiple differential temporal expression, indicating that this protein probably has multiple roles in ontogeny and cell migration.

Transcriptome pyrosequencing of the Antarctic brittle star Ophionotus victoriae.

Brittle stars are included within a whole range of species, which contribute to knowledge in the medically important area of tissue regeneration. All brittle stars regenerate lose limbs, but the rate at which this occurs is highly variable and species-specific. One of the slowest rates of arm regeneration reported so far is that of the Antarctic Ophionotus victoriae. Additionally, O. victoriae also has an unusual delay in the onset of regeneration of about 5months. Both processes are of interest for the areas of regeneration biology and adaptation to cold environments. One method of understanding the details of regeneration events in brittle stars is to characterise the genes involved. In the largest transcriptome study of any ophiuroid to date, we describe the results of mRNA pyrosequencing from pooled samples of regenerating arms of O. victoriae. The sequencing reads resulted in 18,000 assembled contiguous sequences of which 19% were putatively annotated by blast sequence similarity searching. We focus on the identification of major gene families and pathways with potential relevance to the regenerative processes including the Wnt/ß-catenin pathway, Hox genes, the SOX gene family and the TGF beta signalling pathways. These data significantly increase the amount of ophiuroid sequences publicly available and provide candidate transcripts for the further investigation of the unusual regenerative process in this Antarctic ophiuroid.

Xenoturbella bocki exhibits direct development with similarities to Acoelomorpha.

Xenoturbella bocki, a marine animal with a simple body plan, has recently been suggested to be sister group to the Acoelomorpha, together forming the new phylum Xenacoelomorpha. The phylogenetic position of the phylum is still under debate, either as an early branching bilaterian or as a sister group to the Ambulacraria (hemichordates and echinoderms) within the deuterostomes. Although development has been described for several species of Acoelomorpha, little is known about the life cycle of Xenoturbella. Here we report the embryonic stages of Xenoturbella, and show that it is a direct developer without a feeding larval stage. This mode of development is similar to that of the acoelomorphs, supporting the newly proposed phylum Xenacoelomorpha and suggesting that the last common ancestor of the phylum might have been a direct developer.

Pattern of cell proliferation during budding in the colonial ascidian Diplosoma listerianum.

Many invertebrates reproduce asexually by budding, but morphogenesis and the role of cell proliferation in this diverse and nonconserved regeneration-like process are generally poorly understood and particularly little investigated in didemnid ascidians. We here analyzed cell proliferation patterns and telomerase activity during budding in the colonial didemnid ascidian Diplosoma listerianum, with special focus on the thoracic bud where a new brain develops de novo. To help define developmental stages of the thoracic bud, the distribution of acetylated tubulin was also examined. We found extensive cell proliferation in both the thoracic and abdominal buds of D. listerianum as well as higher telomerase activity in bud tissue compared to adult tissues. In the parent adult, proliferation was found in various tissues, but was especially intense in the adult esophagus and epicardial structures that protrude into the proliferating and developing buds, confirming these tissues as the primary source of the cells that form the buds. The neural complex in the thoracic bud forms from a hollow tube that appears to separate into the neural gland and the cerebral ganglion. Whereas most of the bud undergoes proliferation, including the hollow tube and the neural gland, the cerebral ganglion shows little or no proliferation. Pulse-chase labeling experiments indicate that the ganglion, as well as the myocardium, in adult zooids are instead composed of postmitotic cells.

Feeding ecology of Xenoturbella bocki (phylum Xenoturbellida) revealed by genetic barcoding.

The benthic marine worm Xenoturbella is frequently contaminated with molluscan DNA, which had earlier caused confusion resulting in a suggested bivalve relationship. In order to find the source of the contaminant, we have used molluscan sequences derived from Xenoturbella and compared them to barcodes obtained from several individuals of the nonmicroscopic molluscs sharing the same environment as Xenoturbella. Using cytochrome oxidase 1, we found the contaminating sequences to be 98% similar to the bivalve Ennucula tenuis. Using the highly variable D1-D2 region of the large ribosomal subunit in Xenoturbella, we found three distinct species of contaminating molluscs, one of which is 99% similar to the bivalve Abra nitida, one of the most abundant bivalves in the Gullmarsfjord where Xenoturbella was found, and another 99% similar to the bivalve Nucula sulcata. These data clearly show that Xenoturbella only contains molluscan DNA originating from bivalves living in the same environment, refuting former hypotheses of a bivalve relationship. In addition, these data suggest that Xenoturbella feeds specifically on bivalve prey from multiple species, possibly in the form of eggs and larvae.

Induced cell proliferation in putative haematopoietic tissues of the sea star, Asterias rubens (L.).

The coelomic fluid of the echinoderm Asterias rubens possesses large populations of circulating coelomocytes. This study aimed to expand the knowledge about the haematopoietic sources of these cells. Injection of the immune-stimulating molecules lipopolysaccharide (LPS) and concanavalin A (ConA) resulted in an increase in coelomocytes. To investigate if these molecules induce cell proliferation in putative haematopoietic tissues (HPTs), short-term exposure of the substitute nucleotide 5-bromo-2'-deoxyuridine (BrdU) was conducted. Immunohistochemical analysis, using fluorescein-labelled antibodies to trace BrdU, showed pronounced cell division in the coelomic epithelium and axial organ. In the pyloric caeca, not considered as an HPT, proliferation was not detected. BrdU labelling of monolayers of cells obtained by collagenase treatment of coelomic epithelium, axial organ and Tiedemann body revealed induced cell proliferation in response to both LPS and ConA while proliferation of pyloric caeca and circulating coelomocytes remained sparse. By using confocal microscopy it was observed that both the morphology and functional behaviour of cells released from explants of coelomic epithelium showed high similarity to those of circulating phagocytes. It was concluded that the increased coelomocyte numbers observed in response to LPS and ConA were reflected in an induced cell proliferation in coelomic epithelium, axial organ and Tiedemann body, which reinforces the idea that these organs are HPTs and the sources of coelomocyte renewal.