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. J. Exp. Zool. 281:288–304, 1998. © 1998 Wiley‐Liss, Inc.

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 that all four molecules are members of a family of peptides acting as neurotransmitters in echinoderms.

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 [Díaz-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.

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. J. Comp. Neurol. 406:461–475, 1999. © 1999 Wiley‐Liss, Inc.

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