Cell dedifferentiation and epithelial to mesenchymal transitions during intestinal regeneration in H. glaberrima.

BACKGROUND: Determining the type and source of cells involved in regenerative processes has been one of the most important goals of researchers in the field of regeneration biology. We have previously used several cellular markers to characterize the cells involved in the regeneration of the intestine in the sea cucumber Holothuria glaberrima. RESULTS: We have now obtained a monoclonal antibody that labels the mesothelium; the outer layer of the gut wall composed of peritoneocytes and myocytes. Using this antibody we studied the role of this tissue layer in the early stages of intestinal regeneration. We have now shown that the mesothelial cells of the mesentery, specifically the muscle component, undergo dedifferentiation from very early on in the regeneration process. Cell proliferation, on the other hand, increases much later, and mainly takes place in the mesothelium or coelomic epithelium of the regenerating intestinal rudiment. Moreover, we have found that the formation of the intestinal rudiment involves a novel regenerative mechanism where epithelial cells ingress into the connective tissue and acquire mesenchymal phenotypes. CONCLUSIONS: Our results strongly suggest that the dedifferentiating mesothelium provides the initial source of cells for the formation of the intestinal rudiment. At later stages, cell proliferation supplies additional cells necessary for the increase in size of the regenerate. Our data also shows that the mechanism of epithelial to mesenchymal transition provides many of the connective tissue cells found in the regenerating intestine. These results present some new and important information as to the cellular basis of organ regeneration and in particular to the process of regeneration of visceral organs.

Regeneration of the radial nerve cord in the sea cucumber Holothuria glaberrima.

BACKGROUND: Regeneration of neurons and fibers in the mammalian spinal cord has not been plausible, even though extensive studies have been made to understand the restrictive factors involved. New experimental models and strategies are necessary to determine how new nerve cells are generated and how fibers regrow and connect with their targets in adult animals. Non-vertebrate deuterostomes might provide some answers to these questions. Echinoderms, with their amazing regenerative capacities could serve as model systems; however, very few studies have been done to study the regeneration of their nervous system. RESULTS: We have studied nerve cord regeneration in the echinoderm Holothuria glaberrima. These are sea cucumbers or holothurians members of the class Holothuroidea. One radial nerve cord, part of the echinoderm CNS, was completely transected using a scalpel blade. Animals were allowed to heal for up to four weeks (2, 6, 12, 20, and 28 days post-injury) before sacrificed. Tissues were sectioned in a cryostat and changes in the radial nerve cord were analyzed using classical dyes and immunohistochemistry. In addition, the temporal and spatial distribution of cell proliferation and apoptosis was assayed using BrdU incorporation and the TUNEL assay, respectively.We found that H. glaberrima can regenerate its radial nerve cord within a month following transection. The regenerated cord looks amazingly similar in overall morphology and cellular composition to the uninjured cord. The cellular events associated to radial cord regeneration include: (1) outgrowth of nerve fibers from the injured radial cord stumps, (2) intense cellular division in the cord stumps and in the regenerating radial nerve cords, (3) high levels of apoptosis in the RNC adjacent to the injury and within the regenerating cord and (4) an increase in the number of spherule-containing cells. These events are similar to those that occur in other body wall tissues during wound healing and during regeneration of the intestine. CONCLUSION: Our data indicate that holothurians are capable of rapid and complete regeneration of the main component of their CNS. Regeneration involves both the outgrowth of nerve fibers and the formation of neurons. Moreover, the cellular events employed during regeneration are similar to those involved in other regenerative processes, namely wound healing and intestinal regeneration. Thus, holothurians should be viewed as an alternative model where many of the questions regarding nervous system regeneration in deuterostomes could be answered.

Common cellular events occur during wound healing and organ regeneration in the sea cucumber Holothuria glaberrima.

BACKGROUND: All animals possess some type of tissue repair mechanism. In some species, the capacity to repair tissues is limited to the healing of wounds. Other species, such as echinoderms, posses a striking repair capability that can include the replacement of entire organs. It has been reported that some mechanisms, namely extracellular matrix remodeling, appear to occur in most repair processes. However, it remains unclear to what extent the process of organ regeneration, particularly in animals where loss and regeneration of complex structures is a programmed natural event, is similar to wound healing. We have now used the sea cucumber Holothuria glaberrima to address this question. RESULTS: Animals were lesioned by making a 3-5 mm transverse incision between one of the longitudinal muscle pairs along the bodywall. Lesioned tissues included muscle, nerve, water canal and dermis. Animals were allowed to heal for up to four weeks (2, 6, 12, 20, and 28 days post-injury) before sacrificed. Tissues were sectioned in a cryostat and changes in cellular and tissue elements during repair were evaluated using classical dyes, immmuohistochemistry and phalloidin labeling. In addition, the temporal and spatial distribution of cell proliferation in the animals was assayed using BrdU incorporation. We found that cellular events associated with wound healing in H. glaberrima correspond to those previously shown to occur during intestinal regeneration. These include: (1) an increase in the number of spherule-containing cells, (2) remodeling of the extracellular matrix, (3) formation of spindle-like structures that signal dedifferentiation of muscle cells in the area flanking the lesion site and (4) intense cellular division occurring mainly in the coelomic epithelium after the first week of regeneration. CONCLUSION: Our data indicate that H. glaberrima employs analogous cellular mechanisms during wound healing and organ regeneration. Thus, it is possible that regenerative limitations in some organisms are due either to the absence of particular mechanisms associated with repair or the inability of activating the repair process in some tissues or stages.