Participation of the coelomic epithelium of the body wall in muscle regeneration in <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> (Holothuroidea: Aspidochirota)

The cellular sources of regeneration of longitudinal muscles were studied in the holothurian Apostichopus japonicus. An autoradiographic method tracing the distribution of cells labeled with tritiated thymidine (³HT) revealed that the majority of ³HT-cells, which were initially localized in the coelomic epithelium of muscles and the body wall at the beginning of active morphogenesis, were then found in the structure of new muscular bundles during subsequent terms of restoration. Thus, the coelomic epithelium of the body wall participated in the regeneration of muscle tissue concurrently with the coelomic epithelium of muscle, contributing to the recruitment of a pool of myogenic cells.     

Post-autotomy regeneration of respiratory trees in the holothurian Apostichopus japonicus (Holothuroidea, Aspidochirotida)

Specialised respiratory organs, viz. the respiratory trees attached to the dorsal part of the cloaca, are present in most holothurians. These organs evolved within the class Holothuroidea and are absent in other echinoderms. Some holothurian species can regenerate their respiratory trees but others lack this ability. Respiratory trees therefore provide a model for investigating the origin and evolution of repair mechanisms in animals. We conducted a detailed morphological study of the regeneration of respiratory trees after their evisceration in the holothurian Apostichopus japonicus. Regeneration of the respiratory trees occurred rapidly and, on the 15th day after evisceration, their length reached 15–20 mm. Repair involved cells of the coelomic and luminal epithelia of the cloaca. Peritoneocytes and myoepithelial cells behaved differently during regeneration: the peritoneocytes kept their intercellular junctions and migrated as a united layer, whereas groups of myoepithelial cells disaggregated and migrated as individual cells. Although myoepithelial cells did not divide during regeneration, the peritoneocytes proliferated actively. The contractile system of the respiratory trees was assumed to develop during regeneration by the migration of myoepithelial cells from the coelomic epithelium of the cloaca. The luminal epithelium of the respiratory trees formed as a result of dedifferentiation, migration and transformation of cells of the cloaca lining. The mode of regeneration of holothurian respiratory trees is discussed. This work was funded by a grant from the Russian Foundation for Basic Research (project no. 08–04–00284) to I.Y.D. and by a grant from the Far Eastern Branch of the Russian Academy of Sciences and the Russian Foundation for Basic Research (project no. 09–04–98547) to T.T.G.     

Myogenesis during holothurian intestinal regeneration

Echinoderms are well known as being able to regenerate body parts and thus provide excellent models for studying regenerative processes in adult organisms. We are interested in intestinal regeneration in the sea cucumber, Holothuria glaberrima, and focus here on the regeneration of intestinal muscle components. We have used immunohistochemical techniques to describe the formation of the intestinal muscle layers. Myoblasts are first observed within the regenerating structure, adjacent to the coelomic epithelia. Within a few days, these cells acquire muscle markers and form a single cell layer that underlies the epithelia. Animals injected with BrdU at various regeneration stages have been subsequently analyzed for the presence of muscle differentiation markers. BrdU-labeled muscle nuclei are observed in myocytes of 3-week regenerates, showing that these cells originate from proliferating precursors. The peak in muscle precursor proliferation appears to occur during the second week of regeneration. Therefore, new muscle cells in the regenerating intestine originate from precursors that have undergone cell division. Our results suggest that the precursor cells arise from the coelomic epithelia. We also provide a comparative view of muscle regeneration in an echinoderm, a topic of interest in view of the many recent studies of muscle regeneration in vertebrate species. This work was supported by NSF (IBN-0110692) and NIH-MBRS (S06GM08102). We also acknowledge partial support from NIH-RCMI (RRO-3641-01) and the University of Puerto Rico     

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a -/- mutants

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.     

Holotoxin content in the tissues of the holothurian Stichopus japonicus at different seasons of the year and their effect on oocyte maturation

Seasonal variations and distribution of holotoxins in tissues of the sea cucumber Stichopus japonicus have been studied. It was shown that holotoxin content in the ovaries is higher than that in other organs during the breeding season. In the course of a year, the amount of glycosides in other holothurian organs changed a little. It has been found out that high concentrations of glycosides inhibit oocyte maturation in the holothurian Stichopus japonicus.     

The distribution of the Wnt5 protein in the tissues of the holothurian Eupentacta fraudatrix (Djakonov et Baranova, 1958) (Holothuroidea: Dendrochirotida) in the norm and during regeneration

The Wnt5 protein localization in holothurian Eupentacta fraudatrix tissues was examined in the norm and during regeneration. In healthy E. fraudatrix, Wnt5 was found in solitary cells of the hypodermis and in the radial nerve cords. During regeneration, the number of Wnt5 positive cells increased. They were observed in the connective tissue of the body wall and the pharyngeal bulb, in nervous system tissues, the coelomic epithelium, and amoebocytes. The Wnt5 protein may participate in regulating the regeneration in the holothurian E. fraudatrix; it probably modulates cell migration, extracellular matrix reorganization, and neurogenesis.     

Ultrastructure of coelomic lining in echinoderm podia: significance for concepts in the evolution of muscle and peritoneal cells

Summary Ultrastructural data are presented on the histological organization of coelomic lining in the podia of ten species of the five major groups of extant echinoderms. Further evidence of the incorporation of podial retractor muscle cells (myocytes) into a monociliated myoepithelial coelomic lining is provided. In the podia of the crinoid Nemaster rubinginosa and the ophiuroid Ophiophragmus wurdemani as well as in the feeding tentacles of the holothurian Leptosynapta tenuis, coelomic linings are organized as simple myoepithelia consisting of non-contractile peritoneal cells (peritoneocytes) and myocytes. Coelomic linings in the holothurian Thyonella gemmata, the echinoids Eucidaris cf. tribuloides and Lytechinus variegatus, and the asteroids Asterias forbesi and Astropecten sp. are pseudostratified or bipartite pseudostratified myoepithelia consisting of subapical myocytes and apically situated peritoneocytes. The ophiuroid podia of Ophioderma brevispinum and Ophiothrix angulata exhibit transitions from simple myoepithelia to partially pseudostratified epithelia. Intermediate forms between the extremes in myoepithelial organization also occur in the podial lining of single species (e.g. Eucidaris cf. tribuloides). These data supplement recent ultrastructural studies on the podial lining of echinoderms and, in conjunction with published ultrastructural data on the myoepithelial organization of other coelomic linings in echinoderms and in other coelomates, suggest myoepithelial organization of the coelomic lining is a plesiomorph feature in Bilateria.     

Correlating Electrode–Electrolyte Interface and Battery Performance in Hybrid Solid Polymer Electrolyte‐Based Lithium Metal Batteries

Solid polymer electrolytes (SPEs) are desirable in lithium metal batteries (LMBs) since they are nonflammable and show excellent lithium dendrite growth resistance. However, fabricating high performance polymer LMBs is still a grand challenge because of the complex battery system. In this work, a series of tailor‐designed hybrid SPEs are used to prepare LMBs with a LiFePO₄‐based cathode. High performance LMBs with both excellent rate capability and long cycle life are obtained at 60 and 90 °C. The well‐controlled network structure in this series of hybrid SPEs offers a model system to study the relationship between the SPE properties and the LMB performance. It is shown that the cycle life of the polymer LMBs is closely correlated with the SPE–Li interface ionic conductivity, underscoring the importance of the solid electrolyte interface in LMB operation. LMB performance is further correlated with the molecular network structure. It is anticipated that results from this study will shed light on designing SPEs for high performance LMB applications.     

Pharmacological action of the heptapeptide GFSKLYFamide in the muscle of the sea cucumber Holothuria glaberrima (Echinodermata)

The holothurian neuropeptide GFSKLYFamide (GlyPheSerLysLeuTyrPheNH₂), GFSKLYFa, was characterized recently and shown to be present in nerve fibers that apparently innervate various muscle systems. We have studied the potential neurotransmitter role of this peptide by assaying its effects on the contractility of visceral and somatic muscles. GFSKLYFa in nanomolar concentrations induces a relaxation of the muscle tension in the intestine. A similar effect is observed on the longitudinal muscle bands of the body wall of the sea cucumber. The relaxing action of GFSKLYFa is dose dependent suggesting that its action is mediated by receptors present in the muscle cells. In addition, GFSKLYFa induces the relaxation of the acetylcholine contracted intestine. Our investigation provides additional evidence indicating that GFSKLYFa might be a neurotransmitter acting at the neuromuscular junctions of the sea cucumber Holothuria glaberrima.     

Comparative analysis of behavior and proliferative activity in culture of cells of coelomic fluid and of cells of various tissues of the sea star <Emphasis Type="Italic">Asterias rubens</Emphasis> L. Isolated from normal and injured animals

The sources of coelomocytes in Asteroidea are suggested to be the coelomic epithelium, the axial organ, or Tiedemann’s bodies. To study the problem of whether the cells are replenished at the expense of divisions, we analyzed the incorporation of bromodeoxyuridine (BrdU) in vivo into cells from different tissues of the sea star Asterias rubens L. To study differentiation in vitro, methods of isolating and cultivating cells from various tissues were elaborated and an analysis of the behavior and incorporation of BrdU in culture was performed. The reproduced BrdU incorporation was detected in coelomic epithelial cells. The behavior of coelomocytes and the coelomic epithelial cells in culture depended on the time after the injury of the animals in which the cells were isolated, whereas, for the axial organ and Tiedemann’s bodies, no differences were revealed. After 2 months of cultivation, the formation of BrdU-incorporating, colony-like cells with high nuclear-cytoplasmic ratios was characteristic of coelomic epithelial cells. Thus, the most prospective object for studying the processes of A. rubens cell differentiation in vitro seems to be the coelomic epithelium.     

Epidermal ultrastructure of Anoplodium stichopi (Plathelminthes,Dalyellioida), a flatworm endosymbiotic in Stichopus tremulus (Holothurioida)

Summary The epidermal ultrastructure of Anoplodium stichopi Bock 1925 (Platyhelminthes, Dalyellioida, Umagillidae) was studied using transmission and scanning electron microscopy. The species lives in the perivisceral coelom of the aspidochirote holothurian Stichopus tremulus Gunnerus 1767. Two types of cells were observed in the epidermis of A. stichopi: ciliated cuboidal epithelial cells and nonciliated pear-shaped cells. The surface of the ciliated epidermal cells is folded into anastomosing ridges. Numerous coated vesicles are subjacent to the surface folds and mitochondria are abundant just below them. Observations indicate that A. stichopi takes up nutrients pinocytically from the coelomic fluid of the host. The ciliation of A. stichopi is sparse.     

Mitotic Activity in Tissues of the Regenerating Respiratory Tree of the Holothurian Apostichopus japonicus (Holothuroidea,Aspidochirota)

Mitotic activity in the regenerating respiratory tree was studied in the holothurian Apostichopus japonicus after evisceration. Significant proliferation was recorded in the regenerating tissues. The highest number of mitoses was revealed in the inner epithelium, the mitotic index (MI) of which reached 3.44 ± 0.01% on the 10th day of regeneration, which was comparable to MI values of blastema of a regenerating amphibian limb. In the coelomic epithelium the number of dividing cells varied from 0.49% in the upper part (20th day) up to 2.87% in the base (10th day). The proliferative activity gradually decreased in the further course of regeneration. In spite of high MI values, the distribution of mitoses in tissues was even at all stages of regeneration and blastema did not form.     

Localization of the neuropeptide NGIWYamide in the holothurian nervous system and its effects on muscular contraction

NGIWYamide is a peptide recently isolated from the sea cucumber Apostichopus japonicus. It stiffens the connective tissue of the holothurian body wall. Localization of NGIWYamide was investigated by immunohistochemical staining with antiserum raised against NGIWYamide. In holothurian nervous systems NGIWYamide-like immunoreactivity (NGIWYa-LI) was observed in the hyponeural and ectoneural regions of the radial nerve cord, as well as in the circumoral nerve ring, podial nerves, tentacular nerves, the basiepithelial nerve plexus of the intestine and in cellular processes running through the body wall dermis. Labelled nerve fibres from the hyponeural part of the radial nerve running towards the circular muscle and from the podial nerve into the body wall dermis suggest that NGIWYamide controls both muscle and connective tissue. We examined the effect on muscle activity of the sea cucumber. NGIWYamide (10^-7 to 10^-4 M) caused contraction of the longitudinal body wall muscle. Tentacles showed contraction only at a higher dose (10^-4 M). NGIWYamide (10^-4 M) inhibited spontaneous contraction of the intestine.     

Ultrastructure of the coelom in the brachiopod Lingula anatina

The organization of the coelomic system and the ultrastructure of the coelomic lining are used in phylogenetic analysis to establish the relationships between major taxa. Investigation of the anatomy and ultrastructure of the coelomic system in brachiopods, which are poorly studied, can provide answers to fundamental questions about the evolution of the coelom in coelomic bilaterians. In the current study, the organization of the coelom of the lophophore in the brachiopod Lingula anatina was investigated using semithin sectioning, 3D reconstruction, and transmission electron microscopy. The lophophore of L. anatina contains two main compartments: the preoral coelom and the lophophoral coelom. The lining of the preoral coelom consists of ciliated cells. The lophophoral coelom is subdivided into paired coelomic sacs: the large and small sinuses (= canals). The lining of the lophophoral coelom varies in structure and includes monociliate myoepithelium, alternating epithelial and myoepithelial cells, specialized peritoneum and muscle cells, and podocyte‐like cells. Connections between cells of the coelomic lining are provided by adherens junctions, tight‐like junctions, septate junctions, adhesive junctions, and direct cytoplasmic bridges. The structure of the coelomic lining varies greatly in both of the main stems of the Bilateria, that is, in the Protostomia and Deuterostomia. Because of this great variety, the structure of the coelomic lining cannot by itself be used in phylogenetic analysis. At the same time, the ciliated myoepithelium can be considered as the ancestral type of coelomic lining. The many different kinds of junctions between cells of the coelomic lining may help coordinate the functioning of epithelial cells and muscle cells.     

Peritoneal repairing cells: a type of bone marrow derived progenitor cells involved in mesothelial regeneration

The peritoneal mesothelium exhibits a high regenerative ability. Peritoneal regeneration is concomitant with the appearance, in the coelomic cavity, of a free‐floating population of cells whose origin and functions are still under discussion. We have isolated and characterized this cell population and we have studied the process of mesothelial regeneration through flow cytometry and confocal microscopy in a murine model lethally irradiated and reconstituted with GFP‐expressing bone marrow cells. In unoperated control mice, most free cells positive for mesothelin, a mesothelial marker, are green fluorescent protein (GFP). However, 24 hrs after peritoneal damage, free mesothelin⁺/ GFP⁺ cells appear in peritoneal lavages. Cultured lavage peritoneal cells show colocalization of GFP with mesothelial (mesothelin, cytokeratin) and fibroblastic markers. Immunohistochemical staining of the peritoneal wall also revealed colocalization of GFP with mesothelial markers and with procollagen‐1 and smooth muscle α‐actin. This was observed in the injured area as well as in the surrounding not‐injured peritoneal surfaces. These cells, which we herein call peritoneal repairing cells (PRC), are very abundant 1 week after surgery covering both the damaged peritoneal wall and the surrounding uninjured area. However, they become very scarce 1 month later, when the mesothelium has completely healed. We suggest that PRC constitute a type of monocyte‐derived cells, closely related with the tissue‐repairing cells known as ‘fibrocytes’ and specifically involved in peritoneal reparation. Thus, our results constitute a synthesis of the different scenarios hitherto proposed about peritoneal regeneration, particularly recruitment of circulating progenitor cells and adhesion of free‐floating coelomic cells.     

Nephridial development and body cavity formation in <Emphasis Type="Italic">Artemia salina</Emphasis> (Crustacea: Branchiopoda): no evidence for any transitory coelom

The Ecdysozoa-hypothesis on the origin of arthropods questions the homology of segmentation in arthropods, onychophorans, and annelids. The implication of convergent gain of metamery in these groups seems to conflict particularly with the correspondence in the development of serial coelomic cavities and metanephridia. Ultrastructural studies of the mesoderm development in Onychophora revealed that main correspondence with the state in annelids concerns the involvement of epithelial lining cells of the embryonic coelomic cavities in the formation of the visceral and somatic musculature. The significance of this correspondence, however, remained unclear as comparable data on the state in arthropods were still missing. Developmental studies on selected representatives covering all major arthropod subgroups aim to fill in this gap. Data were raised by a combination of transmission electron microscopy and fluorescent stainings of the muscular system and nuclei for the anostracan crustacean Artemia salina. In this species, putative transitory coelomic cavities proved to be absent in all trunk segments. In the second antennal and second maxillary segments small, compact nephridial anlagen develop into a sacculus and excretory duct. The sacculus originates from the terminal cells of the nephridial duct, which is formed in advance. The lumen of the sacculus is inconspicuous in its earliest functional stage and later enlarges to a bulb; it accordingly represents no remnant of any primarily large coelomic cavity. The muscular system is entirely formed prior to and independent of coelomic or nephridial anlagen. Visceral and somatic mesoderm already separate in the caudal body region. Transitory segmental clusters of mesodermal cells are composed of somatic cells only and accordingly represent no “somites”. Our observations overall do not provide any support for the homology of coelomic cavities in annelids and arthropods.     

Filamentous fungi associated with holothurians from the sea of Japan, off the primorye coast of Russia

Holothurians (Holothurioidea, Echinodermata) are known to contain triterpene glycosides, which show antifungal activity. Nevertheless, fungi can be isolated from all organs of holothurians. During 1995-1996, mycelial fungi from several Far-Eastern holothurians--Apostichopus japonicus, Eupentacta fraudatrix, Cucumaria japonica--were collected from the Sea of Japan near the coast of Primorye (Russia) and studied. Twenty-seven species of marine fungi, mostly facultative ones belonging to the mitosporic fungi, were isolated from the holothurians and identified. Fungi isolated from the holothurian surface were more diverse and abundant than those from internal organs and coelomic fluids. Of the holothurians studied, Cucumaria japonica was poorest in abundance and diversity of fungi. The fungi Cladosporium brevicompactum and C. sphaerospermum were common in the holothurian coelom. Because of their high proteolytic activity, these fungi may be pathogenic to holothurians. The detritovorus holothurian A. japonicus was shown to modify the fungal assemblages within the marine bottom sediments.     

A new function of a previously isolated compound that stimulates activation and differentiation of myogenic precursor cells leading to efficient myofiber regeneration and muscle repair

Muscle repair following severe injury is slow and incomplete due to the limited regenerative capacity of muscles comprising the function. In this study, one pure compound structurally corresponding to triterpenoid, which can directly induce the activation, proliferation and maturation of quiescent satellite cells into myocytes in vitro, was isolated from Geum japonicum. The potential effect of this compound on myogenesis was further tested in repair of severe muscle injury. It was found that this compound could significantly stimulate the regenerative potential of the damaged muscle resulting in regeneration of myotubes and myotube bundles time-dependently replacing the damaged muscle tissues. This compound-mediated active regeneration of new myofibers repairing damaged muscles was probably due to its direct action on activation and proliferation of quiescent myogenic precursor cells and enhancement of their maturation into regenerating myotubes, as was demonstrated in our primary myogenic precursor cells culture experiments. The up-regulated expression of endogenous phospho-Akt1 in compound-treated myogenic precursor cells may also contribute to the process of myofiber regeneration and muscle repair probably via promoting myogenic cell survival capacity.