Ultrastructural Features of Gut Regeneration in Five-Month-Old Pentactulae of the Holothurian Eupentacta fraudatrix

Posterior regeneration of the digestive system after bisection was investigated in the anterior halves of the five-month-old pentactulae of the holothurian Eupentacta fraudatrix using electron microscopy. Three stages of gut restoration were distinguished. The first stage is characterized by degradation of the damaged part of the gut followed by wound healing. Active morphogenetic processes (cell proliferation, dedifferentiation, cell migration, and redifferentiation) are observed at the second stage. During the third (final) stage, the ablated parts differentiate in the posterior portion of the intestine. The cells of the gut remnant tissues were shown to be the cell sources of regeneration. Based on both the data available from the literature and the results of our study, the conclusion was drawn that the mechanisms of gut restoration differ significantly in the pentactulae and adults of E. fraudatrix.     

Proteases from the Regenerating Gut of the Holothurian Eupentacta fraudatrix

Four proteases with molecular masses of 132, 58, 53, and 47 kDa were detected in the digestive system of the holothurian Eupentacta fraudatrix. These proteases displayed the gelatinase activity and characteristics of zinc metalloproteinases. The 58 kDa protease had similar protease inhibitor sensitivity to that of mammalian matrix metalloproteinases. Zymographic assay revealed different lytic activities of all four proteases during intestine regeneration in the holothurian. The 132 kDa protease showed the highest activity at the first stage. During morphogenesis (stages 2–4 of regeneration), the highest activity was measured for the 53 and 58 kDa proteases. Inhibition of protease activity exerts a marked effect on regeneration, which was dependent on the time when 1,10-phenanthroline injections commenced. When metalloproteinases were inhibited at the second stage of regeneration, the restoration rates were decreased. However, such an effect proved to be reversible, and when inhibition ceased, the previous rate of regeneration was recovered. When protease activity is inhibited at the first stage, regeneration is completely abolished, and the animals die, suggesting that early activation of the proteases is crucial for triggering the regenerative process in holothurians. The role of the detected proteases in the regeneration processes of holothurians is discussed.     

Ultrastructure of the circumoral nerve ring and the radial nerve cords in holothurians (Echinodermata)

The circumoral nerve ring and the radial nerve cords (RNCs) of Eupentacta fraudatrix and Pseudocnus lubricus (Holothuroidea) were examined as an example of holothurian nervous tissue. The RNC is composed of outer ectoneural and inner hyponeural layers, which are interconnected with one another via short neural bridges. The circumoral nerve ring is purely ectoneural. Both ectoneural and hyponeural components are epithelial tubes with a thick neuroepithelium at one side. A thin ciliated non-neuronal epithelium complements the neuroepithelium to form a tube, thereby enclosing the epineural and hyponeural canals. The whole of the ectoneural and hyponeural subsystems is separated from the surrounding tissue by a continuous basal lamina. The nerve ring and the ectoneural and hyponeural parts of the radial nerves are all neuroepithelia composed of supporting cells and neurons. Supporting cells are interpreted as being glial cells. Based on ultrastructural characters, three types of neurons can be distinguished: (1) putative primary sensory neurons, whose cilium protrudes into the epineural or hyponeural canal; (2) non-ciliated neurons with swollen rough endoplasmic reticulum cisternae; (3) monociliated neurons that are embedded in the trunk of nerve fibers. Different types of synapses occur in the neuropile area. They meet all morphological criteria of classical chemical synapses. Vacuolated cells occur in the neuroepithelium of E. fraudatrix, but are absent in P. lubricus; their function is unknown. The cells of the non-neuronal epithelia that overlie the ectoneural and hyponeural canals are hypothesized to belong to the same cell type as the supporting cells of the neuroepithelium.     

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.     

Heparan Sulfate 6-O-endosulfatases (Sulfs) Coordinate the Wnt Signaling Pathways to Regulate Myoblast Fusion during Skeletal Muscle Regeneration

Skeletal muscle regeneration is mediated by satellite cells (SCs). Upon injury, SCs undergo self-renewal, proliferation, and differentiation into myoblasts followed by myoblast fusion to form new myofibers. We previously showed that the heparan sulfate (HS) 6-O-endosulfatases (Sulf1 and -2) repress FGF signaling to induce SC differentiation during muscle regeneration. Here, we identify a novel role of Sulfs in myoblast fusion using a skeletal muscle-specific Sulf double null (Sulf^SK-DN) mouse. Regenerating Sulf^SK-DN muscles exhibit reduced canonical Wnt signaling and elevated non-canonical Wnt signaling. In addition, we show that Sulfs are required to repress non-canonical Wnt signaling to promote myoblast fusion. Notably, skeletal muscle-relevant non-canonical Wnt ligands lack HS binding capacity, suggesting that Sulfs indirectly repress this pathway. Mechanistically, we show that Sulfs reduce the canonical Wnt-HS binding and regulate colocalization of the co-receptor LRP5 with caveolin3. Therefore, Sulfs may increase the bioavailability of canonical Wnts for Frizzled receptor and LRP5/6 interaction in lipid raft, which may in turn antagonize non-canonical Wnt signaling. Furthermore, changes in subcellular distribution of active focal adhesion kinase (FAK) are associated with the fusion defect of Sulf-deficient myoblasts and upon non-canonical Wnt treatment. Together, our findings uncover a critical role of Sulfs in myoblast fusion by promoting antagonizing canonical Wnt signaling activities against the noncanonical Wnt pathway during skeletal muscle regeneration.     

Dexamethasone treatment in vitro resulted in different responses of two fractions of phagocytes of the holothurian Eupentacta fraudatrix

Echinoderm phagocytes are considered to be analogues to vertebrate macrophages. Previously, the phagocytes of some echinoderm species were divided into two fractions with unclearly identified functional properties. This study aims at modeling the immune response of two phagocyte fractions (P1 and P2) of the holothurian Eupentacta fraudatrix to the synthetic glucocorticoid hormone dexamethasone (Dex) in vitro and at comparison of the effects of such pretreatment on humoral cooperation of each phagocyte fraction with another type of immunocytes, morula cells. During 48-h incubation, Dex (0.1–100 μM) induced apoptosis in a direct (in the P1 fraction) or reverse (in the P2 fraction) concentration-dependent manner. In addition, 100 μM Dex differently affected the cytokin-like substance level in the P1 and P2 phagocyte fractions. Moreover, the supernatants of the Dex(100 μM)-pretreated phagocytes induced opposite changes in the IL-1-like substance level in morula cells. These results indicate a striking functional difference between the two phagocyte fractions. The data obtained provide a new insight into the evolution of macrophage response and into the prospects of the use of in vitro holothurian phagocyte model.     

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.     

Functional morphology of the developing alimentary canal in the holothurian Eupentacta fraudatrix (Holothuroidea, Dendrochirota)

Development of the digestive tract of the holothurian Eupentacta fraudatrix was examined using light and transmission electron microscopy. After the blastopore closes, the gut rudiment loses its connection with the blastoderm and becomes an enclosed, tubular chamber, ending blindly at both ends. The differentiation of the digestive and coelomic epithelia is mainly completed by day 12. Since no transient cell types are observed, this differentiation is definitive. By day 20, the mouth and anal openings appear. The cuticular lining in the anterior part of the gut rudiment has an endodermal origin and differentiates before the mouth is formed. The rest of the gut lining is composed of enterocytes typical of holothuroid intestine. At the early stages of development, mitotic figures are encountered among nonspecialized cells of the gut primordium. In more developed digestive epithelium, vesicular enterocytes are capable of mitotic division. Dividing enterocytes retain secretory vacuoles; thus mitosis occurs in actually differentiated cells. After mouth and anus formation, the oesophagus, stomach, intestine and rectum can be distinguished. In the wall of the stomach, powerful musculature is formed.     

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.     

Dexamethasone Modulation of the Interaction between Two Types of Phagocytes of the Holothurian <Emphasis Type="Italic">Eupentacta fraudatrix</Emphasis> (Djakonov et Baranova, 1958) (Holothuroidea: Dendrochirotida)

This study examines the interaction between two types of phagocytes (P1 and P2) of the holothurian Eupentacta fraudatrix and its in vitro modulation by dexamethasone. Our results indicate that inhibition of apoptosis in P1 phagocytes by P2 phagocytes was accompanied by increased activities of antioxidant enzymes and reduced synthesis of interleukin-1α-like substances. We hypothesize that P1-phagocyte-related effects occurred in response to a high level of hydrogen peroxide produced by P2 phagocytes. The reduced anti-apoptotic effect of P2-phagocyte supernatant during prolonged incubation (24 h) was accompanied by a decline in defense reactions in P1 phagocytes due to depletion of antioxidant enzymes (catalase, glutathione reductase, and glutathione transferase). Inhibition of apoptotis in P1 phagocytes associated with upregulation of antioxidant enzyme defense in response to P2 phagocytes preincubated with dexamethasone (100 µM) indicates that P2 phagocytes affect P1 phagocytes via a ROS-associated mechanism. Thus, our data provide evidence that P1 and P2 phagocytes exhibit their maximum activity at different stages of the immune response, thus causing inhibition of activity in target cells during prolonged exposure. Dexamethasone enhances these effects.     

Phospholipids of the organs and tissues of echinoderms and tunicates from peter the great bay (Sea of Japan)

The phospholipid compositions of organs and tissues were determined in representatives of two phyla of marine invertebrates: Echinodermata [Asteroidea: Aphelasterias japonica (Bell, 1881), Evasterias echinosoma Fisher, 1926, Distolasterias nipon (Doderlein, 1902), Asterias amurensis Lutken, 1871; Echinoidea: Strongylocentrotus intermedius (A. Agassiz, 1863); Holothurioidea: Cucumaria frondosa japonica (Semper, 1868), Eupentacta fraudatrix (Djakonov et Baranova, 1958), Apostichopus japonicus (Selenka, 1867)], and Tunicata [Ascidia: Halocynthia aurantium (Pallas, 1787), H. roretzi (Drasche, 1884), and Styela clava (Herdman, 1881)]. The specificity of phospholipid distribution was shown to be related to the taxonomic position of marine invertebrates and the functional properties of their organs and tissues. Ceramide aminoethylphosphonate was found only in the digestive organs of all holothurians and the starfishes D. nipon and A. amurensis, suggesting its exogenous origin. Phosphatidylglycerol was found in all organs and tissues of the holothurians C. frondosa japonica and E. fraudatrix, as well as in the gastrointestinal tract of ascidians; its origin is unclear.     

Asexual reproduction, evisceration, and regeneration in holothurians (Holothuroidea) from Nha Trang Bay of the South China Sea

Peculiarities of asexual reproduction, evisceration, and regeneration were studied in 13 holothurian species of the orders Dendrochirotida and Aspidochirotida from Nha Trang Bay of the South China Sea. Asexual reproduction by fission has been described for the first time for Cladolabes schmeltzii. It has been shown for the first time that transected C. schmeltzii and Colochirus robustus successfully regenerate not only their anterior, but also their posterior structures. It was found that Pseudocolochirus violaceus was able to regenerate the anterior part of the body and to eject viscera through the anus. The modes of evisceration and regeneration of the gut have been elucidated for ten species of aspidochirotids. It has been shown that the intestine of Holothuria scabra forms from two anlagen rather than from a single one, as previously believed.     

Apoptosis-modulating effect of prostaglandin E<Subscript>2</Subscript> in coelomocytes of holothurian <Emphasis Type="Italic">Eupentacta fraudatrix</Emphasis> depends on the cell antioxidant enzyme status

The effects of prostaglandin PGE₂ on apoptosis and antioxidant enzyme activities were studied in two coelomocyte fractions of holothurian Eupentacta fraudatrix in vitro and in vivo. PGE₂ (10^?8–10^?6M) modulated apoptosis in a time-and concentration-dependent manner in both fractions studied in vitro. In vivo, PGE₂ induced apoptosis at concentrations of 0.1–1 μg/g in the fraction enriched with morula-like cells. Phagocytes were more sensitive to the regulating effect of PGE₂. In this fraction, PGE₂ induced apoptosis at concentrations from 0.01 to 1 μg/g, while PGE₂ at 10 μg/g demonstrated an antiapoptotic effect. In all experiments, apoptosis development was accompanied by a disbalance of the antioxidant enzyme system, primarily, decreased catalase activity.     

Ultrastructure of the Alimentary Canal in Five-Month-Old Pentactulae of the Holothurian Eupentacta fraudatrix

Five-month-old pentactulae (juveniles) of the holothurian Eupentacta fraudatrixpossess a well-developed alimentary canal comprising an esophagus, a stomach, an intestine, and a rectum. The intestine in turn consists of five parts. The esophagus, stomach, and rectum are lined with a cuticular epithelium. The intestinal lining lacks a cuticle and is composed of mainly polyfunctional vesicular enterocytes. Granular enterocytes are less abundant; their cytoplasm contains electron-dense granules, which are probably zymogenic. The gut connective tissue consists of electron-lucent ground substance with collagen fibers and embedded coelomocytes. The gut mesothelium is composed of myoepithelial and peritoneal cells and contains the neurons of the hyponeural nerve plexus.     

Potential vector switching in the evolution of Bursaphelenchus xylophilus group nematodes (Nematoda: Aphelenchoididae)

To show the importance of vector switching of nematodes in the evolution of the Bursaphelenchus xylophilus group, we tested a hypothesis that “Bursaphelenchus doui (or its ancestor) was transferred by Acalolepta fraudatrix, Acalolepta sejuncta, and/or Monochamus subfasciatus (or their ancestral species) from broad‐leaved trees to conifers, switched vectors from these cerambycid beetles to Monochamus beetles in conifers, and then evolved into the common ancestor of Bursaphelenchus mucronatus and B. xylophilus.” We used a simple nematode‐loading method to beetles and produced 20 binary combinations of five B. xylophilus group species and four cerambycid beetle species in the tribe Lamiini. The affinity of the nematodes for the beetles was examined based on phoretic stage formation of the nematodes. Phoretic stages of B. doui appeared in all beetle species examined, namely Acalolepta luxuriosa, Psacothea hilaris, A. fraudatrix, and Monochamus alternatus, although the affinity of the nematode for M. alternatus was weak. This finding indicates that B. doui could switch vectors to conifer‐using Monochamus beetles after transfer by A. fraudatrix from broad‐leaved trees to conifers. We conclude that vector switching of nematodes could have potentially happened during the evolutionary history of the B. xylophilus group.     

Selective Modulation of Wnt Ligands and Their Receptors in Adipose Tissue by Chronic Hyperadiponectinemia

Background

Adiponectin-transgenic mice had many small adipocytes in both subcutaneous and visceral adipose tissues, and showed higher sensitivity to insulin, longer life span, and reduced chronic inflammation. We hypothesized that adiponectin regulates Wnt signaling in adipocytes and thereby modulates adipocyte proliferation and chronic inflammation in adipose tissue.

Materials and Methods

We examined the expression of all Wnt ligands and their receptors and the activity of Wnt signaling pathways in visceral adipose tissue from wild-type mice and two lines of adiponectin-transgenic mice. The effects of adiponectin were also investigated in cultured 3T3-L1 cells.

Results

The Wnt5b, Wnt6, Frizzled 6 (Fzd6), and Fzd9 genes were up-regulated in both lines of transgenic mice, whereas Wnt1, Wnt2, Wnt5a, Wnt9b, Wnt10b, Wnt11, Fzd1, Fzd2, Fzd4, Fzd7, and the Fzd coreceptor low-density-lipoprotein receptor-related protein 6 (Lrp6) were reduced. There was no difference in total β-catenin levels in whole-cell extracts, non-phospho-β-catenin levels in nuclear extracts, or mRNA levels of β-catenin target genes, indicating that hyperadiponectinemia did not affect canonical Wnt signaling. In contrast, phosphorylated calcium/calmodulin-dependent kinase II (p-CaMKII) and phosphorylated Jun N-terminal kinase (p-JNK) were markedly reduced in adipose tissue from the transgenic mice. The adipose tissue of the transgenic mice consisted of many small cells and had increased expression of adiponectin, whereas cyclooxygenase-2 expression was reduced. Wnt5b expression was elevated in preadipocytes of the transgenic mice and decreased in diet-induced obese mice, suggesting a role in adipocyte differentiation. Some Wnt genes, Fzd genes, and p-CaMKII protein were down-regulated in 3T3-L1 cells cultured with a high concentration of adiponectin.

Conclusion

Chronic hyperadiponectinemia selectively modulated the expression of Wnt ligands, Fzd receptors and LRP coreceptors accompanied by the inhibition of the Wnt/Ca²⁺ and JNK signaling pathways, which may be involved in the altered adipocyte cellularity, endogenous adiponectin production, and anti-inflammatory action induced by hyperadiponectinemia.     

Inducible expression of Wnt genes during adult hepatic stem/progenitor cell response

In injured livers where hepatocyte growth is severely limited, facultative hepatic stem/progenitor cells, termed oval cells in rodents, are known to emerge and contribute to the regeneration process. Here, we investigated a possible involvement of Wnt signaling during mouse oval cell response and found significant upregulation of several Wnt genes including Wnt7a, Wnt7b, and Wnt10a. Accordingly, increase of β-catenin protein was observed in oval cell compartments. Pharmacological activation of the canonical Wnt/β-catenin signaling induced proliferation of cultured hepatic stem/progenitor cell lines. These results together implicate the role of Wnt/β-catenin signaling in adult hepatic stem/progenitor cell response.     

Muscle regeneration in the holothurian Stichopus japonicus

The regeneration of longitudinal muscle bands (LMBs) in the sea cucumber Stichopus japonicus was studied using light and electron microscopic and immunocytochemical methods. Previous investigations of holothurian organs showed the presence of some cytoskeletal proteins which were specific for LMBs only. One of them, the 98 KDa protein, was isolated by means of SDS-electrophoresis and used as an antigen to obtain polyclonal antibodies. When tested on paraffin sections of sea cucumber organs, the antibodies were shown to interact only with coelomic epithelial cells covering the LMBs. The antibodies were used to study LMB regeneration after transverse cutting. During regeneration no signs of myocyte dedifferentiation or mitotic division were observed. In the wound region, damaged myocytes degenerated and muscle bundles desintegrated. However, the coelomic epithelial cells dedifferentiated and began to invade the LMB. Just beneath the surface these cells formed clusters (muscle bundle rudiments). The number and size of the clusters gradually increased, the cells lengthened and developed contractile filaments. These observations suggest that new muscle bundles arise from coelomic epithelial cells covering the LMBs. The migration of coelomic epithelial cells into the damaged LMBs and their myogenic transformation are the basic mechanism of holothurian muscle regeneration.