Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1-containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.     

Characteristics of populations of the coelomic fluid and coelomic epithelium cells from the starfish <Emphasis Type="Italic">Asterias rubens</Emphasis> L. able attach to and spread on various substrates

Cultivation is one of the methods of modeling processes occurring in vivo. The success of cultivation, in particular, depends on the choice of substrate. We studied the ability of coelomocytes and coelomic epithelial cells to be attached and spread on fibronectin, laminin, polylysine, and glass. The qualitative composition of heterogeneous populations of coelomocytes and epithelial cells was determined after staining the cells with rhodamine-phalloidin and DAPI, and changes in the composition of populations were evaluated in response to trauma. Seven arbitrary classes of coelomocytes have been identified, three of them being shown to participate in clot formation during primary wound reparation. There was a change in the percentage of these cells attached to specific ligands in response to trauma. In coelomic epithelium, eight arbitrary classes of cells have been identified, two of them being probable candidates for the role of progenitor cells for coelomocytes—coelomocyte-like and small epithelial cells with a high nuclear-cytoplasmic ratio. Enrichment with the small cells in a population of attached coelomic epithelium cells has been revealed when seeding on laminin. Preservation of viability of epithelial cells has been shown when cultured on laminin for 2 months.     

Small coelomic epithelial cells of the starfish Asterias rubens L. that are able to proliferate in vivo and in vitro

Echinoderms, due to their outstanding potential for regeneration, are widely used as experimental models for research in regenerative biology. One of the main problems in this field concerns identification and characterization of cells responsible for the restoration of lost body parts and organs in adult animals. In this study, we analyze the probable candidates for this role in the starfish Asterias rubens L., namely, small coelomic epithelial cells with a high nuclear–cytoplasmic ratio that have the ability to proliferate. These cells are one of several cell types common to the coelomic epithelium (CE) and coelomic fluid (CF). They are analyzed with respect to morphology, proportion in the total cell pool, dynamics after injury and distribution between CE and CF. The results of whole-mount and scanning electron microscopy provide evidence that these small cells occupy a boundary position between CE and CF. Moreover, a novel subpopulation of CE cells is identified that is enriched (up to 50 %) with small epitheliocytes capable of migrating from CE into the CF. As shown in experiments with BrdU incorporation and anti-phospho-histone H3 antibody staining, small epitheliocytes cultured on laminin retain proliferative activity for at least 1 month and can form colony-like aggregates. Two types of small proliferating cells are distinguished by their behavior in culture: some cells remain attached to the substrate and form aggregates, while others detach from the substrate during culturing. The morphology of small epitheliocytes, their proliferative activity in vivo and in vitro and the ability to migrate suggest that they possess certain properties characteristic of stem cells.     

Ultrastructure of coelomic epithelium and coelomocytes of the starfish Asterias rubens L. in norm and after wounding

Samples of coelomic epithelium (CE) and coelomocyte suspension of intact and wounded starfish Asterias rubens L. were studied by electron microscopy. The CE was shown to be composed of three types of cells: flagellar (approximately 60%), secretory (approximately 3%), and myoepithelial (approximately 37%); flagellar and secretory cells form the CE apical surface. Secretory cells are represented by two subtypes, i.e., granular and mucous secretory cells. Myoepithelial cells are located in the basal zone of the epithelium. In 4–5% of cases, adjacent flagellar cells are separated by various sizes of intercellular gaps. These gaps seem to be lacunae left by the flagellar cells after their release into the coelomic cavity. The morphological pattern of the conversion of CE flagellar cells into coelomocytes was characterized. After a moderate wounding used in the present study, no significant structural alterations in the CE organization were revealed. In coelomocyte suspension, small rounded young coelomocytes (approximately 3%) and the larger mature coelomocytes (approximately 97%) were found. On the surface of one of the young coelomocytes, a flagellum was revealed. Surface of the mature coelomocytes forms processes of various size and structure; their cytoplasm contains lysosomes and phagocytic vacuoles of different size. After wounding, a coelomocyte activation was found that consisted of a sharp rise in the number and length of filopodia on their surface, as well as the formation of multicellular aggregates. The complex of ultrastructural data allows it to be suggested that the histogenesis of coelomocytes from CE flagellar cells is a process of cell transdifferentiation.     

An autoradiographic investigation of the proliferative activity of pyloric caeca and gondas of Asterias rubens

The proliferative activity of the pyloric caeca of Asterias rubens was investigated. Autoradiographic experiments using intracoelomically injected (methyl-³H)-thymidine were performed throughout the year and incorporation into pyloric caeca and into gonads was studied. Tritiated thymidine was found to be incorporated mainly in the coelomic lining of both organs. Cell divisions in the coelomic lining may be necessary for the growth of these organs, for the production of coelomocytes or, in the case of the pyloric caeca, for growth of the digestive epithelium. Proliferative activity of the digestive epithelium of the pyloric caeca was only observed in the median duct. It is hypothesized that new cells, arising from mitosis, grow from the median duct to the side lobes and differentiate into storage cells, for example. The existence of a mitosis-inducing or mitosis-stimulating substance is discussed. In the ovaries follicle cells were found to incorporate (methyl-³H)-thymidine; in the testis, proliferation of the germinal epithelium occurred simultaneously in all spermatogenic columns. First, the spermatogonia and then later the spermatocytes became labeled. Absorption of substances from the coelomic fluid is discussed.     

Fine structure of the axial complex of Sphaerechinus granularis (Lam.) (Echinodermata: Echinoidea)

Summary Three regions of the axial complex in Sphaerechinus granularis can be distinguished: 1) The axial organ which protrudes from one side of the axial sinus; the sinus septum which separates the sinus from the body cavity and encloses the stone canal; the pulsating vessel which runs along the inside of the axial organ. 2) The blindly-ending terminal sinus in which the pulsating vessel broadens out to the contractile terminal process. 3) The ampulla of the stone canal which connects the axocoel and water vascular system and which opens out through the madreporite.A single-layered, monociliated coelomic epithelium surrounds all regions of the axial complex. This epithelium contains smooth muscle cells at the contractile areas. Canaliculi, surrounded by basal lamina, are formed through infolding of epithelia; they end blindly in the fluid and connective tissue -matrix of the inner structures.The lacunae of the dorso-ventral mesentery connect the periesophageal and the perianal haemal ring with the axial organ. The axial organ contains many coelomocytes rich in pigment and granules. These coelomocytes are separated into compartments by elastic fibres. Phagocytosis of whole cells and transformational stages of coelomocytes suggest storage and degradation functions. An excretory function via the water vascular system is also suggested.     

The small cells of coelomic fluid and coelomic epithelium isolated from starfish <Emphasis Type="Italic">Asterias rubens</Emphasis> and <Emphasis Type="Italic">Asterias amurensis</Emphasis> (Echinodermata: Asteroidea): Comparative analysis of cell morphology and proliferative activity in vivo and in vitro

Earlier we revealed the probable candidates for the role of Asteroidea stem cells in the starfish Asterias rubens L., small coelomic epithelial cells (SECs-1) with a high nuclear–cytoplasmic ratio that were able to proliferate in vivo and in vitro. To check the existence of a similar cell type in other members of Asteroidea, the small cells in suspensions of coelomic fluid (CF) and coelomic epithelium (CE) of A. amurensis were analyzed with respect to their morphology and proportion in the total cell pool. The morphology of proliferating cells and the proliferative activity of CF and CE cells in vivo and in vitro were studied. The small cells with parameters identical to those of A. rubens SECs-1, were found both in CF and CE of related species. The subpopulation of weakly attached CE cells, highly enriched with SECs-1, was detected. These cells were able to migrate from CE and to proliferate in vivo and in vitro. Additionally, large proliferating cells were described in both starfish. The dynamics of proliferative activity in primary cell cultures of these starfish had some distinctions. Moreover, for the first time, the formation of “crystals”, the potential centres of spiculogenesis, was observed in primary culture of A. amurensis CE cells. The data prove that SECs may fulfil the common functions in two members of Asteroidea.     

Cellular Responses of Congenital Immunity in the Starfish <Emphasis Type="Italic">Asterias rubens</Emphasis>

The work deals with analysis of changes of cellular defense factors in the starfish Asterias rubens in response to injection of human erythrocytes (HE). The number of circulating coelomocytes, dynamics of their production of active oxygen forms, activity of peroxidase, and dynamics of elimination of human hemoglobin from coelomic fluid were estimated before immunization with HE as well as at 6–144 h. The number of coelomocytes was counted in Goryaev chamber, production of active oxygen forms was determined in the test of spontaneous and zymosan-induced reduction of Tetrazolium Nitro Blue, peroxidase activity—in a color enzymatic reaction. Time of human hemoglobin elimination from the coelomic fluid was determined by spectrophotometric method by hemoglobin binding with acetone cyanohydrin with formation of a colored product. It is revealed that injection of human erythrocytes into the starfish Asterias rubens leads to a decrease of the number of coelomocytes in 24–96 h and to an increase of their specific production of active oxygen forms in 96–120 h after the HE injection. In coelomic fluid of Asterias rubens the presence of peroxidase activity is established. The circulation time of human hemoglobin released from erythrocytes in coelomic fluid of these animals does not exceed 24 h. It is suggested that the cellular defense reactions are the major factor of the starfish congenital immunity.__________Translated from Zhurnal Evolyutsionnoi Biokhimii i Fiziologii, Vol. 41, No. 2, 2005, pp. 107–113.Original Russian Text Copyright © 2005 by Kudryavtsev, D’yachkov, Kazakov, Kanaikin, Kharazova, Polevshchikov.     

The characteristic of the coelomic fluid and coelomic epithelium cell populations of starfish Asterias rubens L., capable to attach and spread on various substrates

Cultivation is one of the methods modeling processes occurring in vivo. The success of cultivation, in particular, is defined by a substratum choice. We studied the ability of coelomocytes and coelomic epithelial cells to attach and spread to fibronectin, laminin, polylysine, and glass. Qualitative composition of heterogeneous populations of coelomocytes and epithelial cells was determined after staining the cells with rhodamine-phalloidin and DAPI, and changes in the composition of populations evaluated in response to injury. Seven relative classes of coelomocytes has been identified, three of which has been shown to participate in the formation of clot during primary repair of wounds. There was a change in the proportion of these cells, attached to specific ligands in response to the injury. In coelomic epithelium 8 relative classes of cells has been identified, two of which are likely to be candidates for the role of progenitor cells for coelomocytes--coelomocyte-like and small epithelial cells with high nuclear-cytoplasmic ratio. The enrichment with the small cells in population of attached coelomic epithelium cells has been revealed when seeding on laminin. Continued viability of epithelial cells has been shown when cultured on laminin during 2 months.     

The influence of culture media on embryonic renal collecting duct cell differentiation

Summary During kidney development the embryonic ampullar collecting duct (CD) epithelium changes its function. The capability for nephron induction is lost and the epithelium develops into a heterogeneously composed epithelium consisting of principal and intercalated cells. Part of this development can be mimicked under in vitro conditions, when embryonic collecting duct epithelia are isolated from neonatal rabbit kidneys and kept under perfusion culture. The differentiation pattern is quite different when the embryonic collecting duct epithelia are cultured in standard Iscove’s modified Dulbecco’s medium as compared to medium supplemented with additional NaCl. Thus, the differentiation behavior of embryonic CD epithelia is unexpectedly sensitive. To obtain more information about how much influence the medium has on cell differentiation, we tested medium 199, basal medium Eagle, Williams’ medium E, McCoys 5A medium, and Dulbecco’s modified Eagle medium under serum-free conditions. The experiments show that in general, all of the tested media are suitable for culturing embryonic collecting duct epithelia. According to morphological criteria, there is no difference in morphological epithelial cell preservation. The immunohistochemical data reveal two groups of expressed antigens. Constitutively expressed antigens such as cytokeratin 19, P_CD 9, Na/K ATPase, and laminin are present in all cells of the epithelia independent of the culture media used. In contrast, a group of antigens detected by mab 703, mab 503, and PNA is found only in individual series. Thus, each culture medium produces epithelia with a very specific cell differentiation pattern.     

Air–liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC)

The specific function of the epithelium as critical barrier between the intestinal lumen and the organism’s internal microenvironment is reflected by permanent maintenance of intercellular junctions and cellular polarity. The intestinal epithelial cells are responsible for absorption of nutritional components, facing mechanical stress and a changing oxygen supplementation via blood stream. Oxygen itself can regulate the barrier and the absorptive function of the epithelium. Therefore, we compared the dish cell culture, the transwell-like membrane culture and the oxygen enriched air–liquid interface (ALI) culture. We demonstrated strong influence of the different culture conditions on morphology and function of intestinal porcine epithelial cell lines in vitro. ALI culture resulted in a significant increase in cell number, epithelial cell layer thickness and expression as well as apical localisation of the microvilli-associated protein villin. Remarkable similarities regarding the morphological parameters were observed between ALI cultures and intestinal epithelial cells in vivo. Furthermore, the functional analysis of protein uptake and degradation by the epithelial cells demonstrated the necessity of sufficient oxygen supply as achieved in ALI cultures. Our study is the first report providing marked evidence that optimised oxygen supply using ALI cultures directly affects the morphological differentiation and functional properties of intestinal epithelial cells in vitro.     

Immunolocalization of connexin 43 in the tooth germ of the neonatal rat

Summary Rabbit polyclonal antibodies to amino acids 346–360 of connexin 43, the ‘heart’ gap junction protein, were employed to immunolocalize connexin 43 gap junctions in the neonatal rat molar tooth germ. Connexin 43 appears early in the differentiation of both ectodermally derived and ectomesenchymally derived cells of the developing tooth. Connexin 43 immunoreactivity is present in the epithelial components of the enamel organ, including the area of the proximal and distal junctional complexes of the ameloblast layer, and the stratum intermedium, stellate reticulum and outer enamel epithelium. Secretory odontoblasts and developing alveolar bone also display a pattern of connexin 43 immunostaining. Both the epithelial and ectomesenchymally-derived components of the developing tooth acquire connexin 43 channels in a manner that correlates with cell differentiation. In addition, three regions can be defined by connexin 43 immunostaining: the epithelia of the enamel organ that are derived from the oral epithelium, the odontoblast layer derived from the ectomesenchyme, and the alveolar bone. The results suggest that connexin 43 may provide the mechanism for functional compartmentalization of the tissues associated with tooth formation. Compartmentalization suggested by connexin 43 expression could play important roles in the development and functions of these tissues.     

Fine Structure of the Hepatic Sacculations of Glossobalanus minutus (Enteropneusta,Hemichordata)

Abstract The hepatic region of Glossobalanus minutus is characterized by deep foldings of the dorsal side of the gut epithelium which affect the neighbouring tissues and structures: coelomic spaces, musculature and epidermis. The following cell types of the gut epithelium are described: vacuolated cells, undifferentiated cells, two types of mucous cells and two types of granular secretory cells. The nature and function of the different cell types are discussed. Data on the general ciliation and subepithelial nerve plexus of the gut epithelium are also given, with special mention of a possible neuroendocrine secretion towards the subjacent blood spaces. A well-developed blood sinus (gut sinus) lies between the gut and the visceral peritoneum. The ultrastructural features of the gut epithelium and its close association with the blood sinus point to an absorptive function. The coelomic cavity is reduced to a narrow space limited by two peritoneal sheets (visceral and parietal) of myoepithelial nature. Amoebocyte-like cells (coelomocytes) occur free in the coelomic fluid, and muscular, unicellular bridges are attached to both peritoneal walls across the coelomic space. The dorsal epidermis follows the gut foldings and is formed by flat, overlapping cells. The present observations are compared with previous histological, histochemical and ultrastructural data.     

Structure of the body cavity of the sea spider <Emphasis Type="Italic">Nymphon brevirostre</Emphasis> Hodge, 1863 (Arthropoda: Pycnogonida)

The microscopic anatomy and ultrastructure of the body cavity and adjacent organs in the sea spider Nymphon brevirostre Hodge, 1863 (Pycnogonida, Nymphonidae) were examined by transmission electron microscopy. The longitudinal septa subdividing the body cavity are described: (1) Dohrn’s horizontal septum, (2) lateral heart walls, and (3) paired ventral septa consisting of separate cellular bands. The body cavity is a hemocoel, it has no epithelial lining and is only bordered by a basal lamina. The epidermis, heart, and Dohrn’s septum are not separated from each other by basal laminae and may have a common origin. The cellular bands forming the longitudinal ventral septa are not covered with the basal lamina and presumably derive from cells belonging to the hemocoel. The roles of the morphological structures studied for the circulation of hemolymph are discussed. The gonad lies inside Dohrn’s septum, it is covered with its own basal lamina and surrounded by numerous lacunae of the hemocoel entering the septum. The gonad wall is formed with a single layer of epithelium. The same epithelial cells form the gonad stroma. The gonad cavity is not lined with the basal lamina; muscle cells are present in the gonad wall epithelium, thus rendering the lumen similar to a coelomic cavity. Freely circulating cells of two types are found in the hemocoel: small amebocytes containing electronic-dense granules that are similar to granulocytes of other arthropods, as well as hemocytes with large vacuoles of varying structure that are comparable with plasmatocytes; however some of these may be activated granulocytes.     

Morphological and ultrastructural characterization of sea urchin immune cells

The free circulating coelomocytes in the coelomic cavity of echinoderms are considered to be immune effectors by phagocytosis, encapsulation, cytotoxicity, and by the production of antimicrobial agents. Although echinoderms (especially sea urchin embryo) have been used as a model organisms in biology, no uniform criteria exist for classification of coelomocytes in echinoderms, and few studies have reported about the biological functions of their coelomocytes. Hence, we study the coelomocytes in the echinoid sea urchin, Paracentrotus lividus, and describe their morphological and ultrastructural features using light and transmission electron microscopes. We classify the coelomocytes of P. lividus into red spherule and colorless spherule cells, small cells, vibratile cells, and phagocytic cells; petaloid and filopodial cells. To our knowledge, this is the first report describing ultrastructural details of the coelomocytes of P. lividus. J. Morphol. 276:583–588, 2015. © 2015 Wiley Periodicals, Inc.     

Functional properties of proteins from the coelomic fluid of the wounded sea star Asterias rubens (L)

Impact on viability and adhesion of three protein fractions, separated by size, from the coelomic fluid of wounded Asterias rubens′, was tested on autologous coelomocytes. In addition antimicrobial property of the protein fractions was tested on the Gram-negative bacterium Vibrio parahaemolyticus. All fractions promoted viability and the larger proteins facilitated adhesion of the coelomocytes. The strongest antimicrobial effect was caused by the fraction with the smallest proteins.     

Induction of metamorphosis by thyroid hormone in anuran small intestine cultured organotypically in vitro

Summary We have developed an organ culture system of the anuran small intestine to reproduce in vitro the transition from larval to adult epithelial form which occurs during spontaneous metamorphosis. Tubular fragments isolated from the small intestine ofXenopus laevis tadpoles were slit open and placed on membrane filters in culture dishes. In 60% Leibovitz 15 medium supplemented with 10% charcoal-treated serum, the explants were maintained in good condition for at least 10 days without any morphologic changes. Addition of triiodothyronine (T₃) at a concentration higher than 10⁻⁹ M to the medium could induce cell death of larval epithelial cells, but T₃ alone was not sufficient for proliferation and differentiation of adult epithelial cells. When insulin (5 μg/ml) and cortisol (0.5 μg/ml) besides T₃ were added, the adult cells proliferated and differentiated just as during spontaneous metamorphosis. On Day 5 of cultivation, the adult cells rapidly proliferated to form typical islets, whereas the larval ones rapidly degenerated. At the same time, the connective tissue beneath the epithelium suddenly increased in cell density. These changes correspond to those occurring at the onset of metamorphic climax. By Day 10, the adult cells differentiated into a simple columnar epithelium which possessed the brush border and showed the adult-type lectin-binding pattern. Therefore, the larval epithelium of the small intestine responded to the hormones and transformed into the adult one. This organ culture system may be useful for clarifying the mechanism of the epithelial transition from larval to adult type during metamorphosis.     

Multipotent epithelial cells in the process of regeneration and asexual reproduction in colonial tunicates

The cellular and molecular features of multipotent epithelial cells during regeneration and asexual reproduction in colonial tunicates are described in the present study. The epicardium has been regarded as the endodermal tissue-forming epithelium in the order Enterogona, because only body fragments having the epicardium exhibit the regenerative potential. Epicardial cells in Polycitor proliferus have two peculiar features; they always accompany coelomic undifferentiated cells, and they contain various kinds of organelles in the cytoplasm. During strobilation a large amount of organelles are discarded in the lumen, and then, each tissue-forming cell takes an undifferentiated configuration. Septum cells in the stolon are also multipotent in Enterogona. Free cells with a similar configuration to the septum inhabit the hemocoel. They may provide a pool for epithelial septum cells. At the distal tip of the stolon, septum cells are columnar in shape and apparently undifferentiated. They are the precursor of the stolonial bud. In Pleurogona, the atrial epithelium of endodermal origin is multipotent. In Polyandrocarpa misakiensis, it consists of pigmented squamous cells. The cells have ultrastructurally fine granules in the cytoplasm. During budding, coelomic cells with similar morphology become associated with the atrial epithelium. Then, cells of organ placodes undergo dedifferentiation, enter a cell division cycle, and commence morphogenesis. Retinoic acid-related molecules are involved in this dedifferentiation process of multipotent cells. We conclude that in colonial tunicates two systems support the flexibility of tissue remodeling during regeneration and asexual reproduction; dedifferentiation of epithelial cells and epithelial transformation of coelomic free cells.