Vibrio splendidus flagellin C binds tropomodulin to induce p38 MAPK-mediated p53-dependent coelomocyte apoptosis in Echinodermata.

As a typical pathogen-associated molecular pattern, bacterial flagellin can bind Toll-like receptor 5 and the intracellular NAIP5 receptor component of the NLRC4 inflammasome to induce immune responses in mammals. However, these flagellin receptors are generally poorly understood in lower animal species. In this study, we found that the isolated flagellum of Vibrio splendidus AJ01 destroyed the integrity of the tissue structure of coelomocytes and promoted apoptosis in the sea cucumber Apostichopus japonicus. To further investigate the molecular mechanism, the novel intracellular LRR domain-containing protein tropomodulin (AjTmod) was identified as a protein that interacts with flagellin C (FliC) with a dissociation constant (Kd) of 0.0086 ± 0.33 µM by microscale thermophoresis assay. We show that knockdown of AjTmod also depressed FliC-induced apoptosis of coelomocytes. Further functional analysis with different inhibitor treatments revealed that the interaction between AjTmod and FliC could specifically activate p38 MAPK, but not JNK or ERK MAP kinases. We demonstrate that the transcription factor p38 is then translocated into the nucleus, where it mediates the expression of p53 to induce coelomocyte apoptosis. Our findings provide the first evidence that intracellular AjTmod serves as a novel receptor of FliC and mediates p53-dependent coelomocyte apoptosis by activating the p38 MAPK signaling pathway in Echinodermata.

Morphogenesis and histogenesis during the arm regeneration in a basket star Astrocladus dofleini (Euryalida, Ophiuroidea, Echinodermata).

Basket stars, that is, Ophiuroidea in Echinodermata, exhibit distinctive morphological characteristics with their complicatedly branched arms that can regenerate immediately after mutilation. Although, in brittle stars, that is, ophiuroids with nonbranched arms, the arm regeneration process following accidental trauma or autotomy have been morphologically and histologically observed in several species, few studies have so far been carried out on the regeneration of branched arms in basket stars. In this study, the developmental and morphological features of arm regeneration in Astrocladus dofleini (Gorgonocephalidae, Euryalida, Euryophiurida), one of the most common basket star species in Japanese waters, was anatomically and histologically investigated. Results clearly showed the following phases during the arm regeneration: (a) repair phase, (b) early regenerative phase, (c) intermediate regenerative phase, (d) advanced regenerative Phase I, and (e) advanced regenerative Phase II. The morphogenetic process during the arm regeneration in the basket star showed similar patterns to those of nonbranched arms observed in other ophiuroids. However, differences were also seen between the two ophiuroid types, that is, there were some developmental features specific to the basket star. In the early regenerative phase, branching of coelomic cavities was observed prior to the formation of other tissues, probably inducing the later morphogenesis of branched arms. In addition, hard skeletal ossicles form rapidly at the advanced regenerative Phase II. These developmental features may have led the evolution of bizarre morphologies seen in basket stars, probably contributing to the adaptation to shallow waters from deep-sea habitats.

Are there gap junctions without connexins or pannexins?

BACKGROUND: Gap junctions (GJ) are one of the most common forms of intercellular communication. GJs are assembled from proteins that form channels connecting the cytoplasm of adjacent cells. They are considered to be the main or the only type of intercellular channels and the universal feature of all multicellular animals. Two unrelated protein families are currently considered to be involved in this function, namely, connexins and pannexins (pannexins/innexins). Pannexins were hypothesized to be the universal GJ proteins of multicellular animals, distinct from connexins that are characteristic of chordates only. Here we have revised this supposition by applying growing high throughput sequencing data from diverse metazoan species. RESULTS: Pannexins were found in Chordates, Ctenophores, Cnidarians, and in the most major groups of bilateral protostomes. Yet some metazoans appear to have neither connexins nor pannexins in their genomes. We detected no connexins or pannexins/innexins homologues in representatives of all five classes of echinoderms and their closest relatives hemichordates with available genomic sequences. Despite this, our intracellular recordings demonstrate direct electrical coupling between blastomeres at the 2-cell embryo of the echinoderm (starfish Asterias rubens). In these experiments, carboxyfluorescein fluorescent dye did not diffuse between electrically coupled cells. This excludes the possibility that the observed electrical coupling is mediated by incomplete cytoplasm separation during cleavage. CONCLUSION: Functional GJs are present in representatives of the clade that lack currently recognized GJ protein families. New undiscovered protein families utilized for intercellular channels are predicted. It is possible that the new type(s) of intercellular channels are present in parallel to pannexin and connexin gap junctions in animal groups, other than Echinodermata.

Culturing echinoderm larvae through metamorphosis.

Echinoderms are favored study organisms not only in cell and developmental biology, but also physiology, larval biology, benthic ecology, population biology and paleontology, among other fields. However, many echinoderm embryology labs are not well-equipped to continue to rear the post-embryonic stages that result. This is unfortunate, as such labs are thus unable to address many intriguing biological phenomena, related to their own cell and developmental biology studies, that emerge during larval and juvenile stages. To facilitate broader studies of post-embryonic echinoderms, we provide here our collective experience rearing these organisms, with suggestions to try and pitfalls to avoid. Furthermore, we present information on rearing larvae from small laboratory to large aquaculture scales. Finally, we review taxon-specific approaches to larval rearing through metamorphosis in each of the four most commonly-studied echinoderm classes-asteroids, echinoids, holothuroids and ophiuroids.

Procuring animals and culturing of eggs and embryos.

Echinoderms and especially echinoids have a rich history as model systems for the study of oogenesis, fertilization, and early embryogenesis. The ease of collecting and maintaining adults, as well as in obtaining gametes and culturing large quantities of synchronous embryos, is complemented by the ability to do biochemistry, reverse genetics, embryo manipulations and study gene regulatory networks. The diversity of species and developmental modes as well as unparalleled transparency in early developmental stages also makes echinoderms an excellent system in which to study evolutionary aspects of developmental biology. This chapter provides a practical guide to experimental methods for procuring adults and gametes, achieving synchronous in vitro fertilization, and culturing embryos through early larval stages for several echinoderm species representing four classes (Echinoidea, Asteroidea, Ophiuroidea, and Holothuroidea). We provide specific examples of protocols for obtaining adults and gametes and for culturing embryos of a selected number of species for experimental analysis of their development. The species were chosen to provide breadth across the phylum Echinodermata, as well as to provide practical guidelines for handling some of the more commonly studied species. For each species, we highlight specific advantages, and special note is made of key issues to consider when handling adults, collecting gametes, or setting and maintaining embryo cultures. Finally, information regarding interspecific crosses is provided.

Radial Glia in Echinoderms.

Radial glial cells are crucial in vertebrate neural development and regeneration. It has been recently proposed that this neurogenic cell type might be older than the chordate lineage itself and might have been present in the last common deuterostome ancestor. Here, we summarize the results of recent studies on radial glia in echinoderms, a highly regenerative phylum of marine invertebrates with shared ancestry to chordates. We discuss the involvement of these cells in both homeostatic neurogenesis and post-traumatic neural regeneration, compare the features of radial glia in echinoderms and chordates to each other, and review the molecular mechanisms that control differentiation and plasticity of the echinoderm radial glia. Overall, studies on echinoderm radial glia provide a unique opportunity to understand the fundamental biology of this cell type from evolutionary and comparative perspectives.

Regeneration of the digestive system in the crinoid Himerometra robustipinna occurs by transdifferentiation of neurosecretory-like cells.

The structure and regeneration of the digestive system in the crinoid Himerometra robustipinna (Carpenter, 1881) were studied. The gut comprises a spiral tube forming radial lateral processes, which gives it a five-lobed shape. The digestive tube consists of three segments: esophagus, intestine, and rectum. The epithelia of these segments have different cell compositions. Regeneration of the gut after autotomy of the visceral mass progresses very rapidly. Within 6 h after autotomy, an aggregation consisting of amoebocytes, coelomic epithelial cells and juxtaligamental cells (neurosecretory neurons) forms on the inner surface of the skeletal calyx. At 12 h post-autotomy, transdifferentiation of the juxtaligamental cells starts. At 24 h post-autotomy these cells undergo a mesenchymal-epithelial-like transition, resulting in the formation of the luminal epithelium of the gut. Specialization of the intestinal epithelial cells begins on day 2 post-autotomy. At this stage animals acquire the mouth and anal opening. On day 4 post-autotomy the height of both the enterocytes and the visceral mass gradually increases. Proliferation does not play any noticeable role in gut regeneration. The immersion of animals in a 10-7 M solution of colchicine neither stopped formation of the lost structures nor caused accumulation of mitoses in tissues. Weakly EdU-labeled nuclei were observed in the gut only on day 2 post-autotomy and were not detected at later regeneration stages. Single mitotically dividing cells were recorded during the same period. It is concluded that juxtaligamental cells play a major role in gut regeneration in H. robustipinna. The main mechanisms of morphogenesis are cell migration and transdifferentiation.

Volume regulation of intestinal cells of echinoderms: Putative role of ion transporters (Na(+)/K(+)-ATPase and NKCC).

Echinoderms are exclusively marine osmoconformer invertebrates. Some species occupy the challenging intertidal region. Upon salinity changes, the extracellular osmotic concentration of these animals also varies, exposing tissues and cells to osmotic challenges. Cells and tissues may then respond with volume regulation mechanisms, which involve transport of ions and water into and/or out of the cells, through ion transporters, such as the Na(+)/K(+)-ATPase and NKCC. The goal of this study was to relate the cell volume regulation capacity of echinoderm intestinal cells Na(+)/K(+)-ATPase and NKCC activities, in three echinoderm species: Holothuria grisea, Arbacia lixula, and Echinometra lucunter. Isolated cells of these species displayed some control of their cell volume upon exposure to anisosmotic media (isolated intestinal cells, calcein fluorescence as indicator of volume change), with a distinct higher capacity shown by H. grisea, which did not swell even upon 50% hyposmotic shock. The holothuroid cells showed indirect evidence (effect of furosemide) of the participation of NKCC in this process, with a secretory function, and of a secondary role by the NKA (effect of ouabain). Other mechanisms are probably responsible for this function in the urchins. Variable expression of these transporters, and others not examined here, may to some extent account for the variability in cell volume regulation capacity in echinoderm cells.

Exploring the evolution of marine invertebrate cryopreservation - Landmarks, state of the art and future lines of research.

Lanan (1971) working on oyster sperm and Asahina and Takahashi (1977) on sea urchin sperm and embryos can be considered the pioneers in marine invertebrate cryopreservation. It was from the 90s onwards when the number of references began to increase and diversify not only the range of species of interest but also in the cell types and fields of application. This work is an attempt to summarize the research published on marine invertebrates in a wide variety of journals regarding the development and the applications of marine invertebrate cryopreservation protocols. This paper reviews factors and trends, the obtained results, remaining technical constraints and the immediate future of marine invertebrate cryopreservation.

Echinoderm regeneration: an in vitro approach using the crinoid Antedon mediterranea.

Among echinoderms, crinoids are well known for their remarkable regenerative potential. Regeneration depends mainly on progenitor cells (undifferentiated or differentiated), which migrate and proliferate in the lesion site. The crucial role of the "progenitor" elements involved in the regenerative processes, in terms of cell recruitment, sources, and fate, is a central problem in view of its topical interest and biological implications. The spectacular regenerative potential of crinoids is used to replace lost internal and external organs. In particular, the process of arm regeneration in the feather star Antedon mediterranea is the regeneration model most extensively explored to date. We have addressed the morphological and functional characterization of the cell phenotypes responsible for the arm regenerative processes by using an in vitro approach. This represents the first successful attempt to culture cells involved in crinoid regeneration. A comparison of these results with others from previous in vivo investigations confirms the diverse cell types contributing to regeneration and underscores their involvement in migration, proliferation, and dedifferentiation processes.

Cell cycle arrest and activation of development in marine invertebrate deuterostomes.

Like most metazoans, eggs of echinoderms and tunicates (marine deuterostomes, there is no data for the cephalochordates) arrest awaiting fertilization due to the activity of the Mos/MEK/MAPK cascade and are released from this cell cycle arrest by sperm-triggered Ca2+ signals. Invertebrate deuterostome eggs display mainly three distinct types of cell cycle arrest before fertilization mediated by potentially different cytostatic factors (CSF): one CSF causes arrest during meiotic metaphase I (MI-CSF in tunicates and some starfishes), another CSF likely causes arrest during meiotic metaphase II (amphioxus), and yet another form of CSF causes arrest to occur after meiotic exit during G1 of the first mitotic cycle (G1-CSF). In tunicates and echinoderms these different CSF activities have been shown to rely on the Mos//MAPK pathway for establishment and on Ca2+ signals for their inactivation. Despite these molecular similarities, release of MI-CSF arrest is caused by APC/C activation (to destroy cyclin B) whereas release from G1-CSF is caused by stimulating S phase and the synthesis of cyclins. Further research is needed to understand how both the Mos//MAPK cascade and Ca2+ achieve these tasks in different marine invertebrate deuterostomes. Another conserved feature of eggs is that protein synthesis of specific mRNAs is necessary to proceed through oocyte maturation and to maintain CSF-induced cell cycle arrest. Then activation of development at fertilization is accompanied by an increase in the rate of protein synthesis but the mechanisms involved are still largely unknown in most of the marine deuterostomes. How the sperm-triggered Ca2+ signals cause an increase in protein synthesis has been studied mainly in sea urchin eggs. Here we review these conserved features of eggs (arrest, activation and protein synthesis) focusing on the non-vertebrate deuterostomes.

The elusive role of L-glutamate as an echinoderm neurotransmitter: evidence for its involvement in the control of crinoid arm muscles.

Although l-glutamate is the most widespread excitatory neurotransmitter in vertebrate and invertebrate nervous systems, there is only sparse evidence that it has this role in echinoderms. Following our previous finding that l-glutamate is widely distributed in the arms of the featherstar (crinoid echinoderm) Antedon mediterranea and initiates arm autotomy (defensive detachment), we now provide evidence of glutamatergic involvement in the control of the arm muscles of the same species using immunocytochemical and physiological methods. Immunofluorescence and immunoenzymatic techniques, which employed the same polyclonal antibody against l-glutamate conjugated to glutaraldehyde, revealed a high level of glutamate-like reactivity in the brachial muscles. By recording the mechanical responses of isolated arm pieces, we found that l-glutamate, l-aspartate and elevated [K(+)](o) induced rhythmic muscle contractions, while glycine, γ-aminobutyric acid, adrenaline and acetylcholine had either no, or no consistent, effect. The frequency and duration of the dominant component of the rhythmic contractions indicated that these may be responsible for the rhythmic activity of the arms that occurs during swimming and after autotomy. We conclude that it is highly likely that l-glutamate has at least a neuromodulatory role in the neural pathways controlling the brachial muscles of A. mediterranea.

The mechanically adaptive connective tissue of echinoderms: its potential for bio-innovation in applied technology and ecology.

Echinoderms possess unique connective tissues, called mutable collagenous tissues (MCTs), which undergo nervously mediated, drastic and reversible or irreversible changes in their mechanical properties. Connective tissue mutability influences all aspects of echinoderm biology and is a key-factor in the ecological success of the phylum. Due to their sensitivity to endogenous or exogenous agents, MCTs may be targets for a number of common pollutants, with potentially drastic effects on vital functions. Besides its ecological relevance, MCT represents a topic with relevance to several applied fields. A promising research route looks at MCTs as a source of inspiration for the development of novel biomaterials. This contribution presents a review of MCT biology, which incorporates recent ultrastructural, biomolecular and biochemical analyses carried out in a biotechnological context.

Autofluorescence imaging, an excellent tool for comparative morphology.

Here we present a set of methods for documenting (exo-)morphology by applying autofluorescence imaging. For arthropods, but also for other taxa, autofluorescence imaging combined with composite imaging is a fast documentation method with high-resolution capacities. Compared to conventional micro- and macrophotography, the illumination is much more homogenous, and structures are often better contrasted. Applying different wavelengths to the same object can additionally be used to enhance distinct structures. Autofluorescence imaging can be applied to dried and embedded specimens, but also directly on specimens within their storage liquid. This has an enormous potential for the documentation of rare specimens and especially type specimens without the need of preparation. Also for various fossils, autofluorescence can be used to enhance the contrast between the fossil and the matrix significantly, making even smallest details visible. 'Life-colour' fluorescence especially is identified as a technique with great potential. It provides additional information for which otherwise more complex methods would have to be applied. The complete range of differences and variations between fluorescence macrophotography and different types of fluorescence microscopy techniques are here explored and evaluated in detail. Also future improvements are suggested. In summary, autofluorescence imaging is a powerful, easy and fast-to-apply tool for morphological studies.

Physiological and immunocytochemical evidence that glutamatergic neurotransmission is involved in the activation of arm autotomy in the featherstar Antedon mediterranea (Echinodermata: Crinoidea).

The crinoid echinoderm Antedon mediterranea autotomises its arms at specialised skeletal joints known as syzygies that occur at regular intervals along the length of each arm. Detachment is achieved through the nervously mediated destabilisation of ligament fibres at a particular syzygy. The aim of this investigation was to identify neurotransmitters that are involved in the autotomy response. Physiological experiments were conducted on isolated preparations of syzygial joints, which can be induced to undergo autotomy-like fracture by applying stimulatory agents such as elevated [K(+)](o). Initial experiments with elevated [K(+)](o) showed that the autotomy threshold (the minimum amount of stimulation required to provoke autotomy) is lowest in syzygies at the arm base and rises distally. Of a range of neurotransmitter agonists tested, only l-glutamate invoked syzygial destabilisation, as did its analogues l-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate, but not l-(+)-2-amino-4-phosphonobutyrate (l-AP4) or N-methyl-d-aspartate (NMDA). The implication that l-glutamate stimulates syzygial fracture through AMPA/kainate-like receptors was supported by the finding that the action of l-glutamate was inhibited by the AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Acetylcholine depressed the response of syzygial preparations to l-glutamate, suggesting a possible mechanism by which the autotomy threshold could be varied constitutively and facultatively. An immunocytochemical method employing a polyclonal antibody against l-glutamate conjugated to glutaraldehyde revealed l-glutamate-like immunoreactivity in all components of the putative neural pathway controlling the autotomy reflex, including the epidermis, brachial nerve, syzygial nerves and cellular elements close to the syzygial ligaments. We conclude that it is highly probable that l-glutamate acts as an excitatory neurotransmitter in the activation of arm autotomy in A. mediterranea.

Microscopic anatomy of the digestive system in normal and regenerating specimens of the brittlestar Amphipholis kochii.

The morphology and regeneration of the digestive system of the ophiuroid Amphipholis kochii were investigated. The epithelia of the esophagus and stomach of A. kochii were composed of typical enterocytes and mucous cells. The digestive epithelium of the stomach contained two types of granular secretory cells. After autotomy of the disk, the animals retained the esophagus and a small part of the stomach. The dedifferentiation of enterocytes and mucous cells began on the first day after autotomy. On day 3 the cells formed an anlage of stomach around the mouth opening. Later, the stomach anlage grew as a result of cell proliferation. The opening on the aboral side of the body was closed by day 7. By this time differentiating cells were already observed in the stomach lining. The stomach mesothelium was formed by peritoneocytes and myoepithelial cells, which migrated from other coelomic epithelia of the body. Our study showed that the formation of the digestive system in A. kochii during regeneration depended on cells from the esophagus and the stomach remnant. Both enterocytes and mucous cells were able to dedifferentiate, migrate, and proliferate to give rise to the luminal epithelium. The basic mechanism of stomach formation was epithelial morphogenesis.

Regeneration in crinoids.

Regeneration is a biological phenomenon that occurs in a wide range of animals, and is considered to involve different types of cells including those that are considered to be stem cells. Among the echinoderms, which is a phylum with many regenerating members, crinoids (feather stars and sea lilies) are known to possess high potential of regeneration and are able to regenerate most of their organs. In particular, arm regeneration has been studied using the feather star. During regeneration, coelomocytes and amoebocytes originating from the coelomic canal and the brachial nerve, respectively, migrate to the distal wound area and are involved in the regenerative process. A blastema is formed at the regenerating tip and is derived from migratory amoebocytes. On the other hand, migratory coelomocytes contribute to regenerate the coelomic system. Cells proliferate at the blastema, coelomic canals and brachial nerve. Since the migrating cells differentiate into new structures of the arm, they are considered presumably undifferentiated multipotent stem cells. To deepen our understanding of stem cells in general, we may benefit from an approach from a comparative point of view. Further molecular analyses would increase our knowledge of stem cells in crinoids and allow comparative studies to be possible.

Echinoderm regenerative response as a sensitive ecotoxicological test for the exposure to endocrine disrupters: effects of p,p'DDE and CPA on crinoid arm regeneration.

Echinoderms are valuable test species in marine ecotoxicology and offer a wide range of biological processes appropriate for this approach. Regenerating echinoderms can be regarded as amenable experimental models for testing the effects of exposure to contaminants, particularly endocrine disrupter compounds (EDCs). As regeneration is a typical developmental process, physiologically regulated by humoral mechanisms, it is highly susceptible to the action of pseudo-hormonal contaminants which appear to be obvious candidates for exerting deleterious actions. In our laboratory experiments, selected EDCs suspected for their antiandrogenic action (p,p'-DDE and cyproterone acetate) were tested at low concentrations on regenerating specimens of the crinoid Antedon mediterranea. An integrated approach which combines exposure experiments and different morphological analyses was employed; the obtained results suggest an overall pattern of plausible endocrine disruption in the exposed samples, showing that processes such as regenerative growth, histogenesis, and differentiation are affected by the exposure to the selected compounds. These results confirm that (1) regenerative phenomena of echinoderms can be considered valuable alternative models to assess the effects of exposure to exogenous substances such as EDCs, and (2) these compounds significantly interfere with fundamental processes of developmental physiology (proliferation, differentiation, etc...) plausibly via endocrine alterations. In terms of future prospects, taking into account the increasing need to propose animal models different from vertebrates, echinoderms represent a group on which ecotoxicological studies should be encouraged and specifically addressed.

Apical organs in echinoderm larvae: insights into larval evolution in the Ambulacraria.

The anatomy and cellular organization of serotonergic neurons in the echinoderm apical organ exhibits class-specific features in dipleurula-type (auricularia, bipinnaria) and pluteus-type (ophiopluteus, echinopluteus) larvae. The apical organ forms in association with anterior ciliary structures. Apical organs in dipleurula-type larvae are more similar to each other than to those in either of the pluteus forms. In asteroid bipinnaria and holothuroid auricularia the apical organ spans ciliary band sectors that traverse the anterior-most end of the larvae. The asteroid apical organ also has prominent bilateral ganglia that connect with an apical network of neurites. The simple apical organ of the auricularia is similar to that in the hemichordate tornaria larva. Apical organs in pluteus forms differ markedly. The echinopluteus apical organ is a single structure on the oral hood between the larval arms comprised of two groups of cells joined by a commissure and its cell bodies do not reside in the ciliary band. Ophioplutei have a pair of lateral ganglia associated with the ciliary band of larval arms that may be the ophiuroid apical organ. Comparative anatomy of the serotonergic nervous systems in the dipleurula-type larvae of the Ambulacraria (Echinodermata+Hemichordata) suggests that the apical organ of this deuterostome clade originated as a simple bilaterally symmetric nerve plexus spanning ciliary band sectors at the anterior end of the larva. From this structure, the apical organ has been independently modified in association with the evolution of class-specific larval forms.