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

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

Ultime novit\ue0 nello studio dei tessuti connettivi mutabili (MCTs) : un approccio biochimico e biomolecolare in Paracentrotus lividus

I tessuti connettivi collageni sono ubiquitari nel regno animale e svolgono funzioni di protezione, supporto, immagazzinamento dell'energia elastica e trasferimento di tensione. Gli echinodermi posseggono un peculiare tipo di tessuto connettivo mutabile (MCT), in grado di andare incontro a rapidi cambiamenti delle intrinseche propriet\ue0 meccaniche, mediati dal sistema nervoso, fenomeno noto come mutabilit\ue0. Date queste premesse, gli MCTs possono quindi rappresentare una fonte di ispirazione per biomateriali indirizzati ad esempio all'applicazione biomedica. Le principali componenti extracellulari degli MCTs sono rappresentate da: fibrille di collagene, fibrillina, proteoglicani e glicoproteine, in particolare stiparina e tensilina, che modulano l\u2019aggregazione delle fibrille collagene e le loro possibilit\ue0 di slittamento reciproco, stabilendo legami interfibrillari. Scopo del presente lavoro \ue8 stato mettere a punto un protocollo di estrazione biochimica del collagene dagli MCTs del riccio di mare Paracentrotus lividus, e di purificazione e quantificazione del collagene estratto. Inoltre, dati preliminari sono stati ottenuti con riferimento alla caratterizzazione biomolecolare della tensilina. Il fine ultimo sar\ue0 testare le propriet\ue0 di modulazione da parte della tensilina sullo stato di aggregazione sul collagene estratto a diversi gradi di purezza

Ultrastructural and biochemical characterization of mechanically adaptable collagenous structures in the edible sea urchin Paracentrotus lividus

The viscoelastic properties of vertebrate connective tissues rarely undergo significant changes withinphysiological timescales, the only major exception being the reversible destiffening of the mammalianuterine cervix at the end of pregnancy. In contrast to this, the connective tissues of echinoderms (seaurchins, starfish, sea cucumbers, etc.) can switch reversibly between stiff and compliant conditions intimescales of around a second to minutes. Elucidation of the molecular mechanism underlying suchmutability has implications for the zoological, ecological and evolutionary field. Important informationcould also arise for veterinary and biomedical sciences, particularly regarding the pathological plasti-cization or stiffening of connective tissue structures. In the present investigation we analyzed aspectsof the ultrastructure and biochemistry in two representative models, the compass depressor ligamentand the peristomial membrane of the edible sea urchin Paracentrotus lividus, compared in three differentmechanical states. The results provide further evidence that the mechanical adaptability of echinodermconnective tissues does not necessarily imply changes in the collagen fibrils themselves. The higher gly-cosaminoglycan (GAG) content registered in the peristomial membrane with respect to the compassdepressor ligament suggests a diverse role of these molecules in the two mutable collagenous tissues.The possible involvement of GAG in the mutability phenomenon will need further clarification. Duringthe shift from a compliant to a standard condition, significant changes in GAG content were detected onlyin the compass depressor ligament. Similarities in terms of ultrastructure (collagen fibrillar assembling)and biochemistry (two alpha chains) were found between the two models and mammalian collagen.Nevertheless, differences in collagen immunoreactivity, alpha chain migration on SDS-PAGE and BLASTalignment highlighted the uniqueness of sea urchin collagen with respect to mammalian collagen

New insight into mutable collagenous tissue : work in progress and applied perspectives in Paracentrotus lividus

The mechanically adaptable connective tissue of echinoderms (Mutable Collagenous Tissue, MCT), which can undergo drastic nervously-mediated changes in stiffness, tensile strength and viscosity, represents a promising model for biomaterial design and biomedical applications. MCT could be a source of inspiration for new composite materials whose molecular interactions and structural conformation can be changed in response to external stimuli. MCT is composed of collagen fibrils comparable to those of mammals plus other fibrillar structures, proteoglycans and glycoproteins. According to literature, the extracellular matrix of holothurians includes at least two glycoproteins, stiparin and tensilin, that can modulate the aggregation of collagen fibrils and their capacity for reciprocal sliding. This contribution presents the latest results of a detailed analysis of MCT components in Paracentrotus lividus: focusing on biochemical characterization of the fibrillar components (extraction, purification and quantification) and biomolecular analysis of the glycoprotein components. The final aims will be to confirm the presence and the role of these glycoproteins in echinoids and to manipulate simpler components in order to produce a composite with mutable mechanical properties. In the long term, MCT could provide inspiration for biomimetic materials and offer great potential for economically relevant biotechnological and clinical applications that require the controlled and reversible plasticization and/or stiffening of connective tissue

Echinoderms are valid deuterostome marine invertebrate models to study repair phase events after arm injury

Echinoderms are often subjected to traumatic amputations that damage or remove whole body parts i.e. arms. After such severe injuries, the repair phase must be effective with rapid emergency reaction and re-epithelialisation as well finely regulated extracellular matrix (ECM) remodelling to ensure subsequent arm regeneration. Here, we used the brittle star Amphiura filiformis (Ophiuroidea) and the starfish Echinaster sepositus (Asteroidea) as valid deuterostome marine invertebrate models to study similarities and differences in the repair phase phenomena of these two echinoderm species and discuss them in comparison with those of animals with limited regenerative abilities (i.e. mammals). To achieve this goal, we used an integrated approach based on both microscopy and molecular analyses. We showed that in both echinoderm models, immediately after injury, emergency reaction and re-epithelialisation are extremely rapid and more efficient than those displayed by mammals. The remodelling and the formation of the ECM, mainly collagen, is ensured by delayed activation of ECM genes and protein deposition and, together with absence of fibrosis (i.e. over-deposition of ECM), seem to be advantageous for regeneration-competent animals in comparison to mammals. Overall, we found that the echinoderm species here studied show comparable repair events. The differences between regeneration-competent and non-competent animals suggest that rapid wound closure and delayed ECM deposition are necessary to ensure an effective regeneration of whole lost body parts. Further molecular and functional analyses must be performed to confirm this hypothesis

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

New insights into the mutable collagenous tissue of Paracentrotus lividus : preliminary results

The mechanically adaptable connective tissue of echinoderms (Mutable Collagenous Tissue\u2014MCT), which can undergo drastic nervously-mediated changes in mechanical properties, represents a promising model for biomaterial design and biomedical applications. MCT could be a source of, or an inspiration for, new composite materials whose molecular interactions and structural conformation can be changed in response to external stimuli. MCT is composed mostly of collagen fibrils, comparable to those of mammals, plus a variety of other components, including other fibrillar structures, proteoglycans and glycoproteins. This contribution presents the preliminary results of a detailed analysis of MCT components in the sea-urchin Paracentrotus lividus, focusing on biochemical characterization of the fibrils and biomolecular analysis of the presumptive glycoproteins involved. The final aims will be to confirm the presence and the role of these glycoproteins in echinoids and to manipulate simpler components in order to produce a composite with mutable mechanical properties

Discovery and functional characterization of neuropeptides in crinoid echinoderms.

Neuropeptides are one of the largest and most diverse families of signaling molecules in animals and, accordingly, they regulate many physiological processes and behaviors. Genome and transcriptome sequencing has enabled the identification of genes encoding neuropeptide precursor proteins in species from a growing variety of taxa, including bilaterian and non-bilaterian animals. Of particular interest are deuterostome invertebrates such as the phylum Echinodermata, which occupies a phylogenetic position that has facilitated reconstruction of the evolution of neuropeptide signaling systems in Bilateria. However, our knowledge of neuropeptide signaling in echinoderms is largely based on bioinformatic and experimental analysis of eleutherozoans-Asterozoa (starfish and brittle stars) and Echinozoa (sea urchins and sea cucumbers). Little is known about neuropeptide signaling in crinoids (feather stars and sea lilies), which are a sister clade to the Eleutherozoa. Therefore, we have analyzed transcriptome/genome sequence data from three feather star species, Anneissia japonica, Antedon mediterranea, and Florometra serratissima, to produce the first comprehensive identification of neuropeptide precursors in crinoids. These include representatives of bilaterian neuropeptide precursor families and several predicted crinoid neuropeptide precursors. Using A. mediterranea as an experimental model, we have investigated the expression of selected neuropeptides in larvae (doliolaria), post-metamorphic pentacrinoids and adults, providing new insights into the cellular architecture of crinoid nervous systems. Thus, using mRNA in situ hybridization F-type SALMFamide precursor transcripts were revealed in a previously undescribed population of peptidergic cells located dorso-laterally in doliolaria. Furthermore, using immunohistochemistry a calcitonin-type neuropeptide was revealed in the aboral nerve center, circumoral nerve ring and oral tube feet in pentacrinoids and in the ectoneural and entoneural compartments of the nervous system in adults. Moreover, functional analysis of a vasopressin/oxytocin-type neuropeptide (crinotocin), which is expressed in the brachial nerve of the arms in A. mediterranea, revealed that this peptide causes a dose-dependent change in the mechanical behavior of arm preparations in vitro-the first reported biological action of a neuropeptide in a crinoid. In conclusion, our findings provide new perspectives on neuropeptide signaling in echinoderms and the foundations for further exploration of neuropeptide expression/function in crinoids as a sister clade to eleutherozoan echinoderms