Significant progress has recently been made in our understanding of animal regenerative biology, spurred on by the use of a wider range of model organisms and an increasing ability to use genetic tools in traditional models of regeneration. This progress has begun to delineate differences and similarities in the regenerative capabilities and mechanisms among diverse animal species, and to address some of the key questions about the molecular and cell biology of regeneration. Our expanding knowledge in these areas not only provides insights into animal biology in general, but also has important implications for regenerative medicine and stem-cell biology.

Bridging the regeneration gap: genetic insights from diverse animal models

A Genomic View of the Sea Urchin Nervous System

Autotomy is defined herein as the shedding of a body part, where (1) the loss of the body part is defensive (autotomy helps prevent the whole animal from being compromised and is in response to external stimuli); (2) shearing occurs by an intrinsic mechanism along a breakage plane (there has been selection for certain body parts to be pulled off easily); and (3) the loss is controlled - the animal moves away from the trapped limb, the loss is under some form of central control (neural or hormonal), or the body part is detached quickly. Autotomy (under this defensive definition) has evolved independently for a diverse array of body parts in many taxa; we have summarised available information for over 200 invertebrate species. The advantages of autotomy include escape from entrapment, an effective form of attack, expulsion of an infected body part or in limiting wounding. We discuss how the incidence of autotomy may therefore be correlated with various traits such as limb function, sex differences, other defence mechanisms, habitat disturbance, and sociality. There are also costs associated with autotomy. Short-term costs include loss of a specialised appendage or organ, reduced speed and stability, or even death. Long-term costs include compromised foraging and feeding (often leading to reduced growth), altered anti-predator, competitive or reproductive behaviour, and even defective development. Regenerating lost appendages may also incur significant costs for the individual. We examine the costs and benefits of autotomy, and discuss the evolutionary selective pressures that contribute to the prevalence and effectiveness of autotomy in invertebrates.

Leave it all behind: a taxonomic perspective of autotomy in invertebrates.

Regenerative potential is expressed to a maximum extent in echinoderms. It is a commonphenomenon in all the classes, extensively employed to reconstruct external appendages and internalorgans often subjected to amputation, self-induced or traumatic, rapidly followed by completesuccessful re-growth of the lost parts. Regeneration has been studied in adult individuals as well as inlarvae. In armed echinoderms, regeneration of arms is obviously frequent: in many cases, the detached body fragments can undergo phenomena of partial or total regeneration independently of the donor animal, and, in a few cases (asteroids), the individual autotomised arms can even regenerate to produce new complete adults, offering superb examples of cloning strategies. In the species examined so far most results throw light on aspects related to wound healing, growth, morphogenesis and differentiation, even though in most cases many crucial questions remain unanswered. The present paper provides an overview of the current understanding of the phenomenon and covers the main biological aspects of regeneration giving an idea of the “state of the art” across the phylum in terms of experimental approaches and representative models.

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Regeneration in Echinoderms: repair, regrowth, cloning

Echinoderms are the deuterostome group with the most striking capacity to regenerate lost body parts. In particular, members of the class Holothuroidea are able to regenerate most of their internal organs following a typical evisceration process. Such formation of new viscera in an adult organism provides a unique model to study the process of organogenesis. We have studied this process in the sea cucumber Holothuria glabberrima by describing the spatial and temporal pattern of cellular events that occur during intestine regeneration following chemically induced evisceration. Regeneration begins as a thickening of the mesenteries that supported the autotomized organs to the body wall. The mesenterial thickening consists of tissues where most of the cellular populations found in the normal intestine are already present. However, the cell numbers differ, particularly those of hemocytes and amoebocytes, suggesting that some of these cells play an important role in the formation of the solid rod of hypertrophic mesentery that characterizes the intestinal primordia. The appearance of the luminal epithelium, together with the formation of the lumen, occurs during the second week of regeneration by proliferation and extensive migration of cells from the esophagus and cloacal ends into the thickenings. At this stage all tissue layers are present, but it takes an additional week for them to exhibit the proportions typical of the normal organ. Cell division, as determined by BrdU labeling, mainly occurs in the coelomic epithelia of the hypertrophic mesentery and in the regenerating luminal epithelium. Our study provides evidence that the process of new organ formation in holothurians can be described as an intermediate process showing characteristics of both epimorphic and morphallactic phenomena.

Cellular mechanisms of intestine regeneration in the sea cucumber, Holothuria glaberrima Selenka (Holothuroidea:Echinodermata)

Two peptides, Gly-Phe-Ser-Lys-Leu-Tyr-Phe- NH2 (GFSKLYFamide) and Ser-Gly-Tyr-Ser-Val-Leu-Tyr-Phe- NH2 (SGYSVLYFamide), recently isolated from the sea cucumber Holothuria glaberrima [Diaz-Miranda et al. (1992) Biol. Bull. 182:241–247] represent the first neuropeptides isolated from holothurians. Using an antibody against GFSKLYFa, we describe here the localization and distribution pattern of GFSKLYFa-like immunoreactivity in H. glaberrima, where immunoreactive fibers form a prominent and extensive peptidergic nervous system component. Neuron-like cells and nerve fibers expressing GFSKLYFa-like immunoreactivity are found in the ectoneural and hyponeural divisions of the radial nerve cords as well as in the digestive, haemal, respiratory, and reproductive systems; in the tentacles; and in tube feet. Neuroendocrine-like cells are found in the mucosal layer of the intestine. Ultrastructure immunocytochemical analysis revealed that, in nerve cells and fibers in the serosal layer of the intestine, the immunoreactivity is concentrated in vesicles. The immunoreactive nerve fibers are found mainly within a dense nerve plexus overlying and in close contact with smooth muscle cells of the intestine.



The exclusive expression of GFSKLYFa-like immunoreactivity in neuronal or neuroendocrine tissue together with the close apposition of some fibers to muscle cells suggests that GFSKLYFa acts as a neuromuscular transmitter or neuromodulator in H. glaberrima. The wide occurrence of GFSKLYFa-like immunoreactivity throughout the nervous system of the sea cucumber suggests that GFSKLYFa plays an important role in the control of multiple action systems, including digestion, respiration, circulation, reproduction, and locomotion. © 1995 Wiley-Liss, Inc.

Localization of the heptapeptide GFSKLYFamide in the sea cucumber Holothuria glaberrima (Echinodermata): a light and electron microscopic study.

Two peptides were purified from intestinal extracts of a sea cucumber, Holothuria glaberrima, by high pressure liquid chromatography (HPLC). The peptides were detected by a radioimmunoassay (RIA) based on an antiserum raised to the molluscan peptide, pGlu-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (pQDPFLRFamide). Automated sequencing and mass spectrometry indicate that the isolated peptides are: Gly-Phe-Ser-Lys-Leu-Tyr-Phe-NH2 (GFSKLYFamide) and Ser-Gly-Tyr-Ser-Val-Leu-Tyr-Phe-NH2 (SGYSVLYFamide). These are the first peptides to have been isolated from a member of the echinoderm class Holothuroidea. The antiserum used in the RIA of the peptides was also employed in localizing immunoreactive nerve cells and fibers in the intestine of H. glaberrima. The immunohistochemical results suggest that these peptides might be responsible for the FMRFamide-like immunoreactivity reported earlier. Sequence similarities between GFSKLYFamide, SGYSVLYFamide, and a pair of peptides previously isolated from starfish lead us to propose th...

Characterization of Two Novel Neuropeptides From the Sea Cucumber Holothuria glaberrima.

Among higher metazoans, echinoderms exhibit the most impressive capacity for regeneration. Holothurians, or sea cucumbers, respond to adverse stimuli by autotomizing and ejecting their visceral organs, which are then regenerated. Neuronal fibers and cell bodies are present within the viscera, but previous regeneration studies have not accounted for the nervous component. We used light microscopic immunocytochemistry and ultrastructural studies to describe the regeneration of the enteric nervous system in the sea cucumber Holothuria glaberrima. This study provides evidence that the enteric nervous system of this echinoderm regenerates after evisceration and that in 3-5 weeks the regenerated system is virtually identical to that of noneviscerated animals. The regeneration of the enteric nervous system occurs parallel to the regeneration of other organ components. Nerve fibers and cells are observed within the mesenterial thickenings that give rise to the new intestine and within the internal connective tissue prior to lumen formation. We also used bromodeoxyuridine incorporation to show that proliferation of the neuronal population occurs in the regenerating intestine. The regeneration of the nervous system commands high interest because members of the closely related phylum Chordata either lack or have a very limited capacity to regenerate their nervous system. Thus, holothurians provide a model system to study enteric nervous system regeneration in deuterostomes.

Regeneration of the enteric nervous system in the sea cucumber Holothuria glaberrima.

Echinoderms are one of the most important groups of metazoans from the point of view of evolution, ecology and abundance. Nevertheless, their nervous system has been little studied. Particularly unexplored have been the components of the nervous system that lie outside the ectoneural and hyponeural divisions of the main nerve ring and radial nerve cords. We have gathered information on the nervous components of the digestive tract of echinoderms and demonstrate an unexpected level of complexity in terms of neurons, nerve plexi, their location and neurochemistry. The nervous elements within the digestive system consist of a distinct component of the echinoderm nervous system, termed the enteric nervous system. However, the association between the enteric nervous system and the ectoneural and hyponeural components of the nervous system is not well established. Our findings also emphasize the importance of the large lacunae in the neurobiology of echinoderms, a feature that should be addressed in future studies.

Lucy Díaz-Miranda

Papers

Cellular mechanisms of intestine regeneration in the sea cucumber, Holothuria glaberrima Selenka (Holothuroidea:Echinodermata)

Localization of the heptapeptide GFSKLYFamide in the sea cucumber Holothuria glaberrima (Echinodermata): a light and electron microscopic study.

Characterization of Two Novel Neuropeptides From the Sea Cucumber Holothuria glaberrima.

Regeneration of the enteric nervous system in the sea cucumber Holothuria glaberrima.

The enteric nervous system of echinoderms: unexpected complexity revealed by neurochemical analysis