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

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

Re-exploring epimorphosis vs morphallaxis in echinoderm regeneration

In echinoderms regenerative processes are common in both the adults and larvae and are utilized to replace the loss of different body parts, or for asexual reproduction. Thus echinoderms provide valuable deuterostome models for an integrated approach exploring regeneration from tissue repair to asexual cloning. According to tradition, regeneration involves two alternative basic mechanisms, epimorphosis and morphallaxis. In epimorphosis, new tissues arise from a pool of progenitor cells which are recruited to develop a typical blastema: this discrete proliferation centre contains intrinsic morphogenetic information required to re-pattern the regenerating structure. In morphallaxis, remodelling of the lost part occurs through extensive phenomena of rearrangement/recycling from differentiated tissues, without any blastema formation: only limited and localized proliferation involves cells derived from existing tissues by de-differentiation and/or migration. In echinoderms it was frequently debated if regeneration processes follow morphallaxis or epimorphosis. This basic aspect deserves to be carefully reconsidered, since it implies many crucial questions related to 1) stemness properties of responsible cells (stem cells or reprogrammed cells), 2) activities (proliferation and/or migration), 3) plasticity and differentiation potential (derived cellular phenotypes). Current research is addressed to explore the molecular mechanisms including specific factors involved and differential gene expression. In the present work a comparison of the regeneration mechanisms in representative echinoderm models is provided, with particular reference to crinoids and asteroids: the results, obtained by employing an integrated in vivo and in vitro approach, provide an insight on specificity of mechanisms and processes governing large-scale pattern formation and cell-tissue information signalling. In spite of the schematic epimorphosis vs morphallaxis view, our results show evidence of an unexpected adaptability at the tissue/cellular level and the mechanisms appear rather plastic and largely overlapped to each other, their contribution being different in the different models and even in the same individual according to the specific condition. In addition, in terms of histogenetic potential, different types of tissues appear to be able to give rise to pluripotential cells responsible for development of specific tissues/organs independently of their embryonic origin

Patterns and Cellular Mechanisms of Arm Regeneration in Adult Starfish Echinaster sepositus

Adult echinoderms from each of the five classes exhibit natural, rapid regeneration of entire lost parts following predation or other traumatic events. The asteroids represent one of the echinoderms\u2019 classes used traditionally as experimental models to study limbs regeneration, though very little is known on the cellular mechanisms and patterning. In this study we have chosen \u201cEchinaster sepositus\u201d, the widespread sea star in the Mediterranean as a model with the aim of making a descriptive scenario of the regrowth of missing arm-tip. The regeneration process can be subdivided into three phases: a) a repair phase, which is characterized by wound healing (re-epithelialization) and \u201cedema\u201d formation; b) an early regenerative phase, during which beginning of first differentiation events occurs (skeletogenesis) and c) an advanced regenerative phase characterized by differentiation, morphogenesis and growth of the bud. Our results confirm that regeneration in asteroids is a morphallactic process which usually consists in a rearrangement of the existing tissues through dedifferentiation, differentiation and/or migration of cells in order to regenerate the lost body structures, without a strong contribution of proliferative events. A true blastema is not formed during arm-tip regeneration in this sea star. Therefore, an epimorphollactic process could be involved during regeneration of removed ossicles. This supports the hypothesis of the combination of both processes during regeneration. To confirm such conclusions it\u2019s worthy to use molecular and biochemical approaches

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

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

A microscopic and molecular overview of collagen during echinoderm arm regeneration

Echinoderms are well known for their remarkable regenerative capabilities. The extracellular matrix (ECM) plays a major role during the regenerative process. Collagen is the main ECM component which provides a fundamental structural support; however, little is known about its involvement during regeneration in armed echinoderms. In the present study, adult specimens of the starfish Echinaster sepositus (Retzius, 1783) and of the brittle star Amphiura filiformis (O.F. M\ufcller, 1776) were employed as experimental models to explore from a microscopic anatomy and a molecular perspective the role of collagen in arm regeneration, particularly during the first repair phase, following traumatic amputation (p.a.). Light and electron microscopy were employed to characterize ECM (mainly collagen) during the repair phase. In situ hybridisation and antibody staining for different types of collagen and for a key enzyme of its biosynthesis (prolyl-4-hydroxylase; P4H) were performed at both early and advanced regenerative stages. Our results indicate that immediately after injury the re-epithelialisation of the wound occurs within 24-48 hours p.a. in both E. sepositus and A. filiformis. An oedematous area, composed by a heterogeneous cell population embedded in a disorganized collagen/ECM matrix, is present in starfish 72 hours p.a. Collagen in this area becomes more organised in fibrils and fibres within one week p.a. Instead, in the brittle star an oedema is not present and small collagen fibrils are already present at the early stages under the new epidermis. Not all the A. filiformis selected collagen genes are expressed at early stage, being detectable in different structures at advanced stages, thus indicating diverse spatial and temporal contribution in collagen deposition and maturation throughout the regenerative process. In both starfish and brittle star P4H appears to be expressed in the epidermis at early stage, suggesting the potential role of epidermic cells in collagen biosynthesis. These results seem consistent with the preliminary collagen/P4H antibody stainings performed on both experimental models. Deeper ultrastructural and molecular studies are necessary in order to reach a complete comprehension of the role of collagen and ECM-related molecules during echinoderm arm regeneration