Compared patterns of arm regeneration in different taxa of armed echinoderms

Regeneration is a post-embryonic developmental process common in Metazoa, although it tends to be less widespread in the more complex-bodied phyla. An exception to this rule are echinoderms, which are known for phylum-wide and extremely advanced regenerative abilities, being able to regrow all appendages, and often large parts of the central body and viscera (CANDIA CARNEVALI, 2006). Armed echinoderms (Crinoidea, Asteroidea, and Ophiuroidea) are especially practical models as their arms are easy to amputate, and their proximo-distal extension provides a useful reference point to describe the regenerative processes. Samples of four species from these taxa \u2013 the crinoid Antedon mediterranea, the asteroids Echinaster sepositus and Coscinasterias tenuispina, and the ophiuroid Amphipholis squamata \u2013 were subjected to arm amputation to study the progression of arm regeneration from a morphological point of view by means of different microscopy analyses. Particular attention was given to the \u201caxial structures\u201d, defined as the continuous elements running along the proximo-distal axis of each arm, namely the radial water canal, the radial nerve cord, and the arm coelom, as they are believed to be fundamental for the re-organization of the regenerating arm. The comparison highlighted commonalities and differences of arm regeneration in the different taxa. Distal structures, represented in crinoids by the apical blastema and in asteroids and ophiuroids by the terminal ossicle and tube foot, form very quickly, whereas the proximal region develops later, in proximal-to-distal direction. This is in accordance with previously published models of echinoderm regeneration (MOOI et al., 2005; BEN KHADRA et al., 2018). These similarities suggest that the mechanism of regeneration has ancient origins and is extremely conserved through echinoderm evolution. Within the proximal region, the axial structures themselves develop earlier than the nearby discrete structures (e.g. ossicles and tube feet), and seem to have a crucial role in their organization, providing material and possible signalling molecules for the growing tissue. The cellular component of the nerve grows before any other structure, including its own fibres, thus confirming a primary role of the nervous system in the whole process. Molecular analyses must be combined to morphology data to improve our understanding of similarities and differences of the regenerative process as it occurs in the different echinoderm taxa, as well as in different animal phyla, and to identify related processes in both regeneration-competent and non-competent species. References Ben Khadra Y, Sugni M, Ferrario C, Bonasoro F, Oliveri P, Martinez P, Candia Carnevali MD. 2018. Regeneration in Stellate Echinoderms: Crinoidea, Asteroidea and Ophiuroidea. M. Kloc, J. Z. Kubiak (eds.) Marine Organisms as Model Systems in Biology and Medicine. \ua9Springer International Publishing AG, part of Springer Nature 2018. Chapter 14 Candia Carnevali MD. 2006. Regeneration in Echinoderms: repair, regrowth, cloning. Invertebrate Survival Journal, 3 (1): 64-76 Mooi R, David B, Wray GA. 2005. Arrays in rays: terminal addition in echinoderms and its correlation with gene expression. Evolution & Development, 7 (6): 542-55

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

Nervous system characterization during the development of a basal echinoderm, the feather star Antedon mediterranea

Neural development of echinoderms has always been difficult to interpret, as larval neurons degenerate at metamorphosis and a tripartite nervous system differentiates in the adult. Despite their key phylogenetic position as basal echinoderms, crinoids have been scarcely studied in developmental research. However, since they are the only extant echinoderms retaining the ancestral body plan of the group, crinoids are extremely valuable models to clarify neural evolution in deuterostomes. Antedon mediterranea is a feather star, endemic to the Mediterranean Sea. Its development includes a swimming lecithotrophic larva, the doliolaria, with basiepithelial nerve plexus, and a sessile filter-feeding juvenile, the pentacrinoid, whose nervous system has never been described in detail. Thus, we characterized the nervous system of both these developmental stages by means of immunohistochemistry and, for the first time, in situ hybridization techniques. The results confirmed previous descriptions of doliolaria morphology and revealed that the larval apical organ contains two bilateral clusters of serotonergic cells while GABAergic neurons are localized under the adhesive pit. This suggested that different larval activities (e.g., attachment and metamorphosis) are under the control of different neural populations. In pentacrinoids, the analysis showed the presence of a cholinergic entoneural system while the ectoneural plexus appeared more composite, displaying different neural populations. The expression of three neural-related microRNAs was described for the first time, suggesting that these are evolutionarily conserved also in basal echinoderms. Overall, our results set the stage for future investigations that will reveal new information on echinoderm evo-devo neurobiology

Luminescent conjugates between dinuclear rhenium complexes and 17α-ethynylestradiol: synthesis, photophysical characterization, and cell imaging

Three new luminescent conjugates between dinuclear rhenium complexes and an estradiol, namely E2-Re, are described. The derivatives have the general formula [Re2(\u3bc-Cl)2(CO)6(\u3bc-R-pydz-17\u3b1-ethynylestradiol)] (R-pydz = functionalized 1,2-pyridazine), where the estradiol moiety is covalently bound to the \u3b2 position of the pyridazine ligand. Different synthetic pathways are investigated, including the inverse-type [4 + 2] Diels Alder cycloaddition reaction between the electron poor 1,2,4,5-tetrazine and 17\u3b1-ethynylestradiol for the synthesis of E2-Re1. The three E2-Re conjugates are purified on silica gel and isolated in a spectroscopically pure form in moderate to good yields (28-50%). All the E2-Re conjugates are comprehensively characterized from the spectroscopic and photophysical points of view. Cellular internalization experiments on human MCF-7 and 231 cells are also reported, displaying interesting staining differences depending on the nature of the spacer linking the estradiol unit to the organometallic fragment. Furthermore, the suitability of these conjugates to also stain simple multicellular organisms, i.e. Ciona intestinalis embryos and larvae at different stages of development, is reported here for the first time

Effects of polystyrene microplastics on early stages of two marine invertebrates with different feeding strategies

Nowadays, microplastics represent one of the main threats to marine ecosystems, being able to affect organisms at different stages of their life cycle and at different levels of the food web. Although the presence of plastic debris has been reported in different habitats and the ability to ingest it has been confirmed for different taxa, few studies have been performed to elucidate the effects on survival and development of marine animals. Thus, we explored the effects of different environmental concentrations of polystyrene microbeads on the early stages of two invertebrate species widespread in the Mediterranean shallow waters: the pelagic planktotrophic pluteus larvae of the sea urchin Paracentrotus lividus and the filter-feeding sessile juveniles of the ascidian Ciona robusta. We evaluated the effects on larvae and juvenile development and determined the efficiency of bead ingestion. The feeding stages of both species proved to be extremely efficient in ingesting microplastics. In the presence of microbeads, the metamorphosis of ascidian juveniles was slowed down and development of plutei altered. These results prompted the necessity to monitor the populations of coastal invertebrates since microplastics affect sensitive stages of life cycle and may have consequences on generation recruitment

Bursts of activity in collective cell migration

Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems

Skeletal regeneration in the brittle star Amphiura filiformis

Background: Brittle stars regenerate their whole arms post-amputation. Amphiura filiformis can now be used for molecular characterization of arm regeneration due to the availability of transcriptomic data. Previous work showed that specific developmental transcription factors known to take part in echinoderm skeletogenesis are expressed during adult arm regeneration in A. filiformis; however, the process of skeleton formation remained poorly understood. Here, we present the results of an in-depth microscopic analysis of skeletal morphogenesis during regeneration, using calcein staining, EdU labeling and in situ hybridization. Results: To better compare different samples, we propose a staging system for the early A. filiformis arm regeneration stages based on morphological landmarks identifiable in living animals and supported by histological analysis. We show that the calcified spicules forming the endoskeleton first appear very early during regeneration in the dermal layer of regenerates. These spicules then mature into complex skeletal elements of the differentiated arm during late regeneration. The mesenchymal cells in the dermal area express the skeletal marker genes Afi-c-lectin, Afi-p58b and Afi-p19; however, EdU labeling shows that these dermal cells do not proliferate. Conclusions: A. filiformis arms regenerate through a consistent set of developmental stages using a distalization-intercalation mode, despite variability in regeneration rate. Skeletal elements form in a mesenchymal cell layer that does not proliferate and thus must be supplied from a different source. Our work provides the basis for future cellular and molecular studies of skeleton regeneration in brittle stars

Mutable collagenous tissue: A concept generator for biomimetic materials and devices

Echinoderms (starfish, sea-urchins and their close relations) possess a unique type of collagenous tissue that is innervated by the motor nervous system and whose mechanical properties, such as tensile strength and elastic stiffness, can be altered in a time frame of seconds. Intensive research on echinoderm ‘mutable collagenous tissue’ (MCT) began over 50 years ago, and over 20 years ago, MCT first inspired a biomimetic design. MCT, and sea-cucumber dermis in particular, is now a major source of ideas for the development of new mechanically adaptable materials and devices with applications in diverse areas including biomedical science, chemical engineering and robotics. In this review, after an up-to-date account of present knowledge of the structural, physiological and molecular adaptations of MCT and the mechanisms responsible for its variable tensile properties, we focus on MCT as a concept generator surveying biomimetic systems inspired by MCT biology, showing that these include both bio-derived developments (same function, analogous operating principles) and technology-derived developments (same function, different operating principles), and suggest a strategy for the further exploitation of this promising biological resource

Fundamental aspects of arm repair phase in two echinoderm models

Regeneration is a post-embryonic developmental process that ensures complete morphological and functional restoration of lost body parts. The repair phase is a key step for the effectiveness of the subsequent regenerative process: in vertebrates, efficient re-epithelialisation, rapid inflammatory/immune response and post-injury tissue remodelling are fundamental aspects for the success of this phase, their impairment leading to an inhibition or total prevention of regeneration. Among deuterostomes, echinoderms display a unique combination of striking regenerative abilities and diversity of useful experimental models, although still largely unexplored. Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here used to comparatively investigate the main repair phase events after injury as well as the presence and expression of immune system and extracellular matrix (i.e. collagen) molecules using both microscopy and molecular tools. Our results showed that emergency reaction and re-epithelialisation are similar in both echinoderm models, being faster and more effective than in mammals. Moreover, in comparison to the latter, both echinoderms showed delayed and less abundant collagen deposition at the wound site (absence of fibrosis). The gene expression patterns of molecules related to the immune response, such as Ese-fib-like (starfishes) and Afi-ficolin (brittle stars), were described for the first time during echinoderm regeneration providing promising starting points to investigate the immune system role in these regeneration models. Overall, the similarities in repair events and timing within the echinoderms and the differences with what has been reported in mammals suggest that effective repair processes in echinoderms play an important role for their subsequent ability to regenerate. Targeted molecular and functional analyses will shed light on the evolution of these abilities in the deuterostomian lineage

Gli echinodermi come modelli per lo studio di Distruttori Endocrini: un approccio integrato

Gli echinodermi rappresentano dei validi modelli sperimentali nell\u2019ambito dell\u2019ecotossicologia marina. Essi infatti sono organismi bentonici a diretto contatto con potenziali fonti di xenobiotici e offrono un ampio range di processi biologici utili per questo tipo di approccio. In questo lavoro sono presentati i risultati pi\uf9 significativi ottenuti nel nostro laboratorio utilizzando questi organismi come modelli per lo studio degli effetti di potenziali Distruttori Endocrini (ED). Sono stati presi in esame due diverse specie di echinodermi, il riccio di mare Paracentrotus lividus e il crinoide Antedon mediterranea, e due diversi aspetti della loro fisiologia, la biologia riproduttiva e lo sviluppo rigenerativo, rispettivamente. Esemplari adulti di entrambe le specie sono stati esposti a diversi ED, potenzialmente in grado di interferire con gli ormoni sessuali. Le concentrazioni utilizzate erano comparabili a quelle ambientali. La ricerca si \ue8 basata su un approccio multidisciplinare che ha previsto analisi morfologiche, chimiche e biochimiche. I risultati ottenuti hanno evidenziato la capacit\ue0 di questi composti di interferire con diversi parametri quali la crescita rigenerativa, il pattern istologico e il diametro delle uova prodotte, sebbene lo specifico meccanismo d\u2019azione sia ancora da chiarire. Infatti, nel caso di P. lividus, la somministrazione diretta di ormoni sessuali, quali l\u2019estradiolo, non produce effetti significativi sulla biologia riproduttiva dell\u2019animale. Nel complesso i risultati ottenuti sottolineano sia l\u2019importanza di utilizzare modelli alternativi nei test ecotossicologici sia la necessit\ue0 di approfondire maggiormente la fisiologia di base degli organismi modello