Ultrastructural and molecular analysis of the origin and differentiation of cells mediating brittle star skeletal regeneration.

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

Methodology for Whole Mount and Fluorescent RNA In Situ Hybridization in Echinoderms: Single, Double, and Beyond.

Identifying the location of a specific RNA in a cell, tissue, or embryo is essential to understand its function. Here we use echinoderm embryos to demonstrate the power of fluorescence in situ RNA hybridizations to localize sites of specific RNA accumulation in whole mount embryo applications. We add to this technology the use of various probe-labeling technologies to colabel multiple RNAs in one application and we describe protocols for incorporating immunofluorescence approaches to maximize the information obtained in situ. We offer alternatives for these protocols and troubleshooting advice to identify steps in which the procedure may have failed. Overall, echinoderms are wonderfully suited for these technologies, and these protocols are applicable to a wide range of cells, tissues, and embryos.

The structural origins of brittle star arm kinematics: An integrated tomographic, additive manufacturing, and parametric modeling-based approach.

Brittle stars are known for the high flexibility of their arms, a characteristic required for locomotion, food grasping, and for holding onto a great diversity of substrates. Their high agility is facilitated by the numerous discrete skeletal elements (ossicles) running through the center of each arm and embedded in the skin. While much has been learned regarding the structural diversity of these ossicles, which are important characters for taxonomic purposes, their impact on the arms' range of motion, by contrast, is poorly understood. In the present study, we set out to investigate how ossicle morphology and skeletal organization affect the flexibility of brittle star arms. Here, we present the results of an in-depth analysis of three brittle star species (Ophioplocus esmarki, Ophiopteris papillosa, and Ophiothrix spiculata), chosen for their different ranges of motion, as well as spine size and orientation. Using an integrated approach that combines behavioral studies with parametric modeling, additive manufacturing, micro-computed tomography, scanning electron microscopy, and finite element simulations, we present a high-throughput workflow that provides a fundamental understanding of 3D structure-kinematic relationships in brittle star skeletal systems.

Functional Morphology of the Arm Spine Joint and Adjacent Structures of the Brittlestar Ophiocomina nigra (Echinodermata: Ophiuroidea).

The skeletal morphology of the arm spine joint of the brittlestar Ophiocomina nigra was examined by scanning electron microscopy and the associated epidermis, connective tissue structures, juxtaligamental system and muscle by optical and transmission electron microscopy. The behaviour of spines in living animals was observed and two experiments were conducted to establish if the spine ligament is mutable collagenous tissue: these determined (1) if animals could detach spines to which plastic tags had been attached and (2) if the extension under constant load of isolated joint preparations was affected by high potassium stimulation. The articulation normally operates as a flexible joint in which the articular surfaces are separated by compliant connective tissue. The articular surfaces comprise a reniform apposition and peg-in-socket mechanical stop, and function primarily to stabilise spines in the erect position. Erect spines can be completely immobilised, which depends on the ligament having mutable tensile properties, as was inferred from the ability of animals to detach tagged spines and the responsiveness of isolated joint preparations to high potassium. The epidermis surrounding the joint has circumferential constrictions that facilitate compression folding and unfolding when the spine is inclined. The interarticular connective tissue is an acellular meshwork of collagen fibril bundles and may serve to reduce frictional forces between the articular surfaces. The ligament consists of parallel bundles of collagen fibrils and 7-14 nm microfibrils. Its passive elastic recoil contributes to the re-erection of inclined spines. The ligament is permeated by cell processes containing large dense-core vesicles, which belong to two types of juxtaligamental cells, one of which is probably peptidergic. The spine muscle consists of obliquely striated myocytes that are linked to the skeleton by extensions of their basement membranes. Muscle contraction may serve mainly to complete the process of spine erection by ensuring close contact between the articular surfaces.

Paedomorphosis as an Evolutionary Driving Force: Insights from Deep-Sea Brittle Stars.

Heterochronic development has been proposed to have played an important role in the evolution of echinoderms. In the class Ophiuroidea, paedomorphosis (retention of juvenile characters into adulthood) has been documented in the families Ophiuridae and Ophiolepididae but not been investigated on a broader taxonomic scale. Historical errors, confusing juvenile stages with paedomorphic species, show the difficulties in correctly identifying the effects of heterochrony on development and evolution. This study presents a detailed analysis of 40 species with morphologies showing various degrees of juvenile appearance in late ontogeny. They are compared to a range of early ontogenetic stages from paedomorphic and non-paedomorphic species. Both quantitative and qualitative measurements are taken and analysed. The results suggest that strongly paedomorphic species are usually larger than other species at comparable developmental stage. The findings support recent notions of polyphyletic origin of the families Ophiuridae and Ophiolepididae. The importance of paedomorphosis and its correct recognition for the practice of taxonomy and phylogeny are emphasized.

When Ontogeny Matters: A New Japanese Species of Brittle Star Illustrates the Importance of Considering both Adult and Juvenile Characters in Taxonomic Practice.

Current taxonomy offers numerous approaches and methods for species delimitation and description. However, most of them are based on the adult characters and rarely suggest a dynamic representation of developmental transformations of taxonomically important features. Here we show how the underestimation of ontogenetic changes may result in long term lack of recognition of a new species of one of the most common ophiacanthid brittle stars (Echinodermata: Ophiuroidea) from the North Pacific. Based on vast material collected predominantly by various Japanese expeditions in the course of more than 50 years, and thorough study of appropriate type material, we revise the complex of three common species of the ophiuroid genus Ophiacantha which have been persistently confused with each other. The present study thus reveals the previously unrecognized new species Ophiacantha kokusai sp.nov. which is commonly distributed off the Pacific coast of Japan. The new species shows developmental differentiation from the closely related species Ophiacantha rhachophora H. L. Clark, 1911 and retains clearly expressed early juvenile features in the adult morphology. Another species, Ophiacantha clypeata Kyte, 1977, which had been separated from O. rhachophora, is in turn shown to be just a juvenile stage of another North Pacific species, Ophiacantha trachybactra H.L. Clark, 1911. For every species, detailed morphological data from both adult and juvenile specimens based on scanning electron microscopy are presented. A special grinding method showing complex internal features has been utilized for the first time. For all three species in this complex, a clear bathymetric differentiation is revealed: O. rhachophora predominantly inhabits shallow waters, 0-250 m, the new species O. kokusai lives deeper, at 250-600 m, and the third species, O. trachybactra, is found at 500-2,000 m. The present case clearly highlights the importance of considering developmental transformations, not only for a limited number of model organisms, but as part of the taxonomic process.

Microlens arrays in the complex visual system of Cretaceous echinoderms.

It has long been assumed that photosensitivity in echinoderms is mainly related to diffuse photoreception mediated by photosensitive regions embedded within the dermis. Recent studies, however, have shown that some extant echinoderms may also display modified ossicles with microlenses acting as sophisticated photosensory organs. Thanks to their remarkable properties, these calcitic microlenses serve as an inspiration for scientists across various disciplines among which bio-inspired engineering. However, the evolutionary origins of these microlenses remain obscure. Here we provide microstructural evidence showing that analogous spherical calcitic lenses had been acquired in some brittle stars and starfish of Poland by the Late Cretaceous (Campanian, ~79 Ma). Specimens from Poland described here had a highly developed visual system similar to that of modern forms. We suggest that such an optimization of echinoderm skeletons for both mechanical and optical purposes reflects escalation-related adaptation to increased predation pressure during the so-called Mesozoic Marine Revolution.

Micro- to nanostructure and geochemistry of extant crinoidal echinoderm skeletons.

This paper reports the results of micro- to nanostructural and geochemical analyses of calcitic skeletons from extant deep-sea stalked crinoids. Fine-scale (SEM, FESEM, AFM) observations show that the crinoid skeleton is composed of carbonate nanograins, about 20-100 nm in diameter, which are partly separated by what appears to be a few nm thick organic layers. Sub-micrometre-scale geochemical mapping of crinoid ossicles using a NanoSIMS ion microprobe, combined with synchrotron high-spatial-resolution X-ray micro-fluorescence (µ-XRF) maps and X-ray absorption near-edge structure spectroscopy (XANES) show that high Mg concentration in the central region of the stereom bars correlates with the distribution of S-sulphate, which is often associated with sulphated polysaccharides in biocarbonates. These data are consistent with biomineralization models suggesting a close association between organic components (including sulphated polysaccharides) and Mg ions. Additionally, geochemical analyses (NanoSIMS, energy dispersive spectroscopy) reveal that significant variations in Mg occur at many levels: within a single stereom trabecula, within a single ossicle and within a skeleton of a single animal. Together, these data suggest that physiological factors play an important role in controlling Mg content in crinoid skeletons and that great care should be taken when using their skeletons to reconstruct, for example, palaeotemperatures and Mg/Ca palaeo-variations of the ocean.

Staging of regeneration process of an arm of the feather star Oxycomanthus japonicus focusing on the oral-aboral boundary.

Crinoids have strong regenerative capability and rapidly restore their lost body parts such as arms. We observed the regeneration process of arms of the feather star (stalkless crinoid), Oxycomanthus japonicus, and divided the process into 10 stages. We clarify the position at which the oral and aboral epidermis adhere in wound closure and track the oral-aboral boundary in the regenerate during the entire process of regeneration. We suggest that the concepts of distalization and intercalation, which are proposed to understand animal regeneration integrally, are also applicable to arm regeneration of the feather star. In addition, we clarify that pinnules, appendages extending from the sides of an arm, start to grow in the oral region of the regenerating arm even though a complete pinnule has an oral-aboral axis. The mode of morphogenesis of pinnules in arm regeneration suggests that the oral region functions as the primary patterning tissue for pinnules.

Development and growth of the feather star Oxycomanthus japonicus to sexual maturity.

Crinoids, including feather stars, are the most basal group among extant echinoderm classes and share a basic body plan. In spite of their importance for evolutionary developmental study, information on the development of crinoids has been limited, because there are not many species whose spawning season is known, and artificial spawning is impossible. Therefore, it is not easy to obtain fertilized eggs of crinoids. We have observed the spawning and development of the feather star Oxycomanthus japonicus for 7 years. We have established a cultivation system that has enabled us to culture large numbers of O. japonicus from eggs through to sexually mature adults. In the present study, we show that (1) individuals take 2 years to reach sexual maturity; (2) the skeleton of the theca of a stalked juvenile consists of five orals, five basals, five radials, five infrabasals, and an anal plate; and (3) the onset of spawning has shifted by about two weeks since 60 years ago. Our cultivation system can provide enough embryos, larvae, juveniles, and adults for further experiments, extending the possibilities for crinoid research.

Surface microtopographies of tropical sea stars: lack of an efficient physical defence mechanism against fouling.

The role of surface topography as a defence against fouling in tropical sea stars was investigated. The sea stars Linckia laevigata, Fromia indica, Cryptasterina pentagona and Archaster typicus are not fouled and have paxillae (modified ossicles with a median vertical pillar) on their aboral surfaces, which varied in diameter, height and distance depending on species and position on the aboral surface, providing unique and complex surface microtopographies for each species. The surfaces of the sea stars L. laevigata, F. indica and A. typicus were moderately wettable, with their mean seawater contact angles, calculated from captive bubble measurements, being 60.1 degrees, 70.3 degrees and 57.3 degrees, respectively. The seawater contact angle of C. pentagona could not be measured. To evaluate the effectiveness of the surface microtopographies in deterring the settlement of fouling organisms, field experiments with resin replicas of the four sea star species were conducted at three sites around Townsville, Australia, for 8 weeks during the dry and wet seasons. The fouling community and total fouling cover did not differ significantly between replicas of L. laevigata, F. indica, C. pentagona, A. typicus and control surfaces at any site during the dry season. Significant differences between fouling communities on the replicas of the sea stars and control surfaces were detected at two sites during the wet season. However, these differences were transitory, and the total fouling cover did not differ significantly between replicas of sea stars and control surfaces at two of the three sites. In contrast to recent literature on the effects of biofouling control by natural surfaces in the marine environment, the surface microtopographies of tropical sea stars alone were not effective in deterring the settlement and growth of fouling organisms.

Sclerites in different tissues of Mediterranean Echinodermata.

The presence of sclerites in different mesodermal tissues was studied in thirteen species of Mediterranean echinoderms representing the five living classes. In the species of Asteroidea and Crinoidea examined, sclerites were present only in specific tissues. In the species of Ophiuroidea and, especially, Echinoidea and Holothuroidea examined, sclerites were present in almost every tissue, but with considerable variation among species and tissues. Data on sclerite shape, size, and relative abundance (measured as tissue abundance) are presented. The data support the possible functional role of sclerites as reinforcing structures, as well as their possible use as taxonomic characters. Finally, the need of more studies to elucidate the ontogenetic and phylogenetic implications of sclerites is discussed.

Organization of the coelomic lining and a juxtaposed nerve plexus in the suckered tube feet of Parastichopus californicus (Echinodermata: Holothuroida).

The coelomic lining of the water-vascular canal in a suckered tube foot from the sea cucumber, Parastichopus californicus, is a pseudostratified myoepithelium consisting of flagellated adluminal cells and myofilament-bearing retractor cells. The bodies of adluminal cells flank the water-vascular canal and send basal processes between the underlying retractor cells to confront the podial connective tissue. Retractor cells have a contractile apparatus of unregistered thick and thin myofilaments. The contractile apparatus is confined to the medullary sarcoplasm and oriented parallel to the primary axis of a tube foot. The bodies and processes of retractor cells intermingle with the basal processes of adluminal cells at the basal lamina of the coelomic lining. A ganglionated nerve plexus in the podial connective tissue approximates the basal lamina. Neuronal connectives link the ganglia to one another and to the nerve plexus in deep sectors of the podial epidermis. External laminae enveloping the ganglia and connectives in the podial connective tissue are continuous with the basal lamina of the epidermis. The adventitial nerve plexus, since it merges with the epidermal nerve plexus, is a component of the ectoneural division of the echinoderm nervous system.

Echinoderm adhesive secretions: from experimental characterization to biotechnological applications.

Adhesion is a way of life in echinoderms. Indeed, all the species belonging to this phylum use adhesive secretions extensively for various vital functions. According to the species or to the developmental stage considered, different adhesive systems may be recognized. (1) The tube feet or podia are organs involved in attachment to the substratum, locomotion, feeding or burrowing. Their temporary adhesion relies on a duo-gland adhesive system resorting to both adhesive and de-adhesive secretions. (2) The larval adhesive organs allow temporary attachment of larvae during settlement and strong fixation during metamorphosis. (3) The Cuvierian tubules are sticky defence organs occurring in some holothuroid species. Their efficacy is based on the instantaneous release of a quick-setting adhesive. All these systems rely on different types of adhesion and therefore differ in the way they operate, in their structure and in the composition of their adhesive. In addition to fundamental interests in echinoderm bioadhesives, a substantial impetus behind understanding these adhesives are the potential technological applications that can be derived from their knowledge. These applications cover two broad fields of applied research: design of water-resistant adhesives and development of new antifouling strategies. In this context, echinoderm adhesives could offer novel features or performance characteristics for biotechnological applications. For example, the rapidly attaching adhesive of Cuvierian tubules, the releasable adhesive of tube feet or the powerful adhesive of asteroid larvae could each be useful to address particular bioadhesion problems.

Calcitic microlenses as part of the photoreceptor system in brittlestars.

Photosensitivity in most echinoderms has been attributed to 'diffuse' dermal receptors. Here we report that certain single calcite crystals used by brittlestars for skeletal construction are also a component of specialized photosensory organs, conceivably with the function of a compound eye. The analysis of arm ossicles in Ophiocoma showed that in light-sensitive species, the periphery of the labyrinthic calcitic skeleton extends into a regular array of spherical microstructures that have a characteristic double-lens design. These structures are absent in light-indifferent species. Photolithographic experiments in which a photoresist film was illuminated through the lens array showed selective exposure of the photoresist under the lens centres. These results provide experimental evidence that the microlenses are optical elements that guide and focus the light inside the tissue. The estimated focal distance (4-7 micrometer below the lenses) coincides with the location of nerve bundles-the presumed primary photoreceptors. The lens array is designed to minimize spherical aberration and birefringence and to detect light from a particular direction. The optical performance is further optimized by phototropic chromatophores that regulate the dose of illumination reaching the receptors. These structures represent an example of a multifunctional biomaterial that fulfills both mechanical and optical functions.

Microscopic overview of crinoid regeneration.

Crinoids are well known for their striking regenerative potential and can rapidly and completely regenerate arms lost following self-induced or traumatic amputation. Thus they provide a valuable experimental model for investigation of the regenerative process from the macroscopic to the molecular level. In these last years we have studied in detail the overall process of arm regeneration in the comatulid Antedon mediterranea. This phenomenon can be described on the whole as a typical blastemal regeneration in which new structures develop from migratory pluripotential, actively proliferating cells in the presence of presumptive regulatory factors. The overall process can be subdivided into three main phases: a repair phase, an early regenerative phase, and an advanced regenerative phase, whose crucial aspects are related to common fundamental mechanisms such as cell migration and proliferation, intervention of stem cells and/or dedifferentiated cells, contribution of putative growth factors, particularly in terms of specific neurally derived factors, and mechanisms of pattern formation. This article focuses on the main aspects of the phenomenon and gives a brief account of the most recent and relevant results. Our approach employs classical methods of light (LM) and electron (TEM and SEM) microscopy, immunocytochemistry, and histofluorescence on experimentally induced arm regenerations of standard or abnormal type obtained in significantly different experimental conditions, including extreme mutilations (explants) or exposure to pseudo-estrogenic environmental contamination.

Cirri of the stalked crinoid Metacrinus rotundus: neural elements and the effect of cholinergic agonists on mechanical properties.

Sea lilies are enigmatic animals due to their scarcity and their biology is comparatively neglected. Cirri, arranged in whorls of five along the sea lily stalk, anchor and support the animal. They consist of ossicles interconnected by collagenous ligaments and by a central canal. Cirri have a well-developed nervous system but lack muscular cells. A light and electron microscopic study was performed to clarify the morphology of the nervous system of the cirri. Two cellular networks were found, one of neuron-like cells and one of cells filled with bullet-shaped organelles. Both networks ramify throughout the cirral ossicles up to the interossicle ligaments. Mechanical tests were performed to analyse the influence of cholinergic agonists on the mechanical properties of these ligaments. In the tests, the cirral ligaments softened after the application of acetylcholine, muscarinic agonists and nicotinic agonists. The reaction time to muscarinic agonists was much slower than to acetylcholine and nicotinic agonists. At low concentrations, muscarinic agonists caused active development of force. No reaction to stimuli was observed in anaesthetized cirri. The data clearly establish the existence of catch connective tissue which can change its mechanical properties under nervous control mediated via nerves with cholinergic receptors. The possible sources of the observed force production are discussed and it is concluded that active contraction of collagenous ligaments causes movement of cirri.

Symbioses in Amphipholis squamata (Echinodermata, Ophiuroidea, Amphiuridae): geographical variation of infestation and effect of symbionts on the host's light production.

Populations of the polychromatic and bioluminescent species Amphipholis squamata from eight locations were examined for internal and external symbionts. At three locations (two in the United Kingdom and one in Papua New Guinea), no symbionts were present, while four species were recovered from the remaining locations: Cancerilla tubulata and Parachordeumium amphiurae (copepods), Rhopalura ophiocomae (orthonectid) and an undescribed species of rhabdocoel turbellarian. No ophiuroid individual hosted more than one symbiont species, despite the presence of two or more within a population. Symbiont presence and prevalence varied with location, and with colour variety, but with no apparent pattern or trends. Light-production characteristics of the host were affected by the presence of all symbionts except C. tubulata. These effects, however, did not vary between colour varieties or between geographical locations, but were specific to the symbiont species: the presence of P. amphiurae resulted in enhanced intensity of light production, while that of R. ophiocomae and the turbellarian species resulted in reduced intensity. The kinetics of light production (time until maximum output) were altered only by the presence of the turbellarian. Changes in the light-production characteristics are discussed in relation to morphological, energetical and physiological effects of the symbioses.