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.

An integrated view of asteroid regeneration: tissues, cells and molecules.

The potential for repairing and replacing cells, tissues, organs and body parts is considered a primitive attribute of life shared by all the organisms, even though it may be expressed to a different extent and which is essential for the survival of both individual and whole species. The ability to regenerate is particularly evident and widespread within invertebrates. In spite of the wide availability of experimental models, regeneration has been comprehensively explored in only a few animal systems (i.e., hydrozoans, planarians, urodeles) leaving many other animal groups unexplored. The regenerative potential finds its maximum expression in echinoderms. Among echinoderm classes, asteroids offer an impressive range of experimental models in which to study arm regeneration at different levels. Many studies have been recently carried out in order to understand the regenerative mechanisms in asteroids and the overall morphological processes have been well documented in different starfish species, such as Asterias rubens, Leptasterias hexactis and Echinaster sepositus. In contrast, very little is known about the molecular mechanisms that control regeneration development and patterning in these models. The origin and the fate of cells involved in the regenerative process remain a matter of debate and clear insights will require the use of complementary molecular and proteomic approaches to study this problem. Here, we review the current knowledge regarding the cellular, proteomic and molecular aspects of asteroid regeneration.

Wound repair during arm regeneration in the red starfish Echinaster sepositus.

Starfish can regenerate entire arms following their loss by both autotomic and traumatic amputation. Although the overall regenerative process has been studied several times in different asteroid species, there is still a considerable gap of knowledge as far as the detailed aspects of the repair phase at tissue and cellular level are concerned, particularly in post-traumatic regeneration. The present work is focused on the arm regeneration model in the Mediterranean red starfish Echinaster sepositus; to describe the early cellular mechanisms of arm regeneration following traumatic amputation, different microscopy techniques were employed. In E. sepositus, the repair phase was characterized by prompt wound healing by a syncytial network of phagocytes and re-epithelialisation followed by a localized subepidermal oedematous area formation. Scattered and apparently undifferentiated cells, intermixed with numerous phagocytes, were frequently found in the wound area during these first stages of regeneration and extensive dedifferentiation phenomena were seen at the level of the stump, particularly in the muscle bundles. A true localized blastema did not form. Our results confirm that regeneration in asteroids mainly relies on morphallactic processes, consisting in extensive rearrangement of the existing tissues which contribute to the new tissues through cell dedifferentiation, redifferentiation, and/or migration.

Re-growth, morphogenesis, and differentiation during starfish arm regeneration.

The red starfish Echinaster sepositus is an excellent model for studying arm regeneration processes following traumatic amputation. The initial repair phase was described in a previous paper in terms of the early cicatrisation phenomena, and tissue and cell involvement. In this work, we attempt to provide a further comprehensive description of the later regenerative stages in this species. Here, we present the results of a detailed microscopic and submicroscopic investigation of the long regenerative phase, which can be subdivided into two subphases: early and advanced regenerative phases. The early regenerative phase (1-6 weeks p.a.) is characterized by tissue rearrangement, morphogenetic processes and initial differentiation events (mainly neurogenesis and skeletogenesis). The advanced regenerative phase (after 6 weeks p.a.) is characterized by further differentiation processes (early myogenesis), and obvious morphogenesis and re-growth of the regenerate. As in other starfish, the regenerative process in E. sepositus is relatively slow in comparison with that of crinoids and many ophiuroids, which is usually interpreted as resulting mainly from size-related aspects and of the more conspicuous involvement of morphallactic processes. Light and electron microscopy analyses suggest that some of the amputated structures, such as muscles, are not able to replace their missing parts by directly re-growing them from the remaining tissues, whereas others tissues, such as the skeleton and the radial nerve cord, appear to undergo direct re-growth. The overall process is in agreement with the distalization-intercalation model proposed by Agata and co-workers. Further experiments are needed to confirm this hypothesis.

Homeobox genes expressed during echinoderm arm regeneration.

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

Studying Echinodermata Arm Explant Regeneration Using Echinaster sepositus.

Echinoderms are marine invertebrate deuterostomes known for their amazing regenerative abilities throughout all life stages. Though some species can undergo whole-body regeneration (WBR), others exhibit more restricted regenerative capabilities. Asteroidea (starfish) comprise one of the few echinoderm taxa capable of undergoing WBR. Indeed, some starfish species can restore all tissues and organs not only during larval stages, but also from arm fragments as adults. Arm explants have been used to study cells, tissues and genes involved in starfish regeneration. Here, we describe methods for obtaining and studying regeneration of arm explants in starfish, in particular animal collection and husbandry, preparation of arm explants, regeneration tests, microscopic anatomy techniques (including transmission electron microscopy, TEM) used to analyze the regenerating explant tissues and cells plus a downstream RNA extraction protocol needed for subsequent molecular investigations.

Regeneration in Stellate Echinoderms: Crinoidea, Asteroidea and Ophiuroidea.

Reparative regeneration is defined as the replacement of lost adult body parts and is a phenomenon widespread yet highly variable among animals. This raises the question of which key cellular and molecular mechanisms have to be implemented in order to efficiently and correctly replace entire body parts in any animal. To address this question, different studies using an integrated cellular and functional genomic approach to study regeneration in stellate echinoderms (crinoids, asteroids and ophiuroids) had been carried out over the last few years. The phylum Echinodermata is recognized for the striking regeneration potential shown by the members of its different clades. Indeed, stellate echinoderms are considered among the most useful and tractable experimental models for carrying comprehensive studies focused on ecological, developmental and evolutionary aspects. Moreover, most of them are tractable in the laboratory and, thus, should allow us to understand the underlying mechanisms, cellular and molecular, which are involved. Here, a comprehensive analysis of the cellular/histological components of the regenerative process in crinoids, asteroids and ophiuroids is described and compared. However, though this knowledge provided us with some clear insights into the global distribution of cell types at different times, it did not explain us how the recruited cells are specified (and from which precursors) over time and where are they located in the animal. The precise answer to these queries needs the incorporation of molecular approaches, both descriptive and functional. Yet, the molecular studies in stellate echinoderms are still limited to characterization of some gene families and protein factors involved in arm regeneration but, at present, have not shed light on most of the basic mechanisms. In this context, further studies are needed specifically to understand the role of regulatory factors and their spatio-temporal deployment in the growing arms. A focus on developing functional tools over the next few years should be of fundamental importance.

Low genetic diversity and recent demographic expansion in the red starfish Echinaster sepositus (Retzius 1816).

Understanding the phylogeography and genetic structure of populations and the processes responsible of patterns therein is crucial for evaluating the vulnerability of marine species and developing management strategies. In this study, we explore how past climatic events and ongoing oceanographic and demographic processes have shaped the genetic structure and diversity of the Atlanto-Mediterranean red starfish Echinaster sepositus. The species is relatively abundant in some areas of the Mediterranean Sea, but some populations have dramatically decreased over recent years due to direct extraction for ornamental aquariums and souvenir industries. Analyses across most of the distribution range of the species based on the mitochondrial cytochrome c oxidase subunit I gene and eight microsatellite loci revealed very low intraspecific genetic diversity. The species showed a weak genetic structure within marine basins despite the a priori low dispersal potential of its lecithotrophic larva. Our results also revealed a very recent demographic expansion across the distribution range of the species. The genetic data presented here indicate that the species might be highly vulnerable, due to its low intraspecific genetic diversity.