Telomerase activity is not related to life history stage in the jellyfish Cassiopea sp.

The polyp (scyphistoma) of the jellyfish Cassiopea sp. can be maintained in culture for a long time, as polyps repeatedly reproduce asexually via formation of vegetative buds or propagules. The medusa, which is the sexually reproducing stage, typically has a relatively short life span. As a first step to understand the difference in life spans of the polyp and medusa stages of Cassiopea sp., we measured telomerase activity in different life cycle stages. We found telomerase activity in tissues of aposymbiotic polyps and propagules and symbiotic ephyrae (newly budded medusae) and adult medusae. No significant difference in telomerase activity was found between polyps and the bell region of the medusae. The cloned elongation products of the stretch PCR contained the TTAGGG repeats suggesting that the jellyfish has the ‘vertebrate’ telomere motif (TTAGGG)_n. This is the first study to show that somatic tissues of both polyp and medusa stages of a cnidarian had telomerase activity. Telomerase activity in somatic tissues may be related to the presence of multipotent interstitial cells and high regenerative capacity of cnidarians.     

Polypengeneration und Entwicklungsgeschichte vonEucheilota maculata (Thecata-Leptomedusae)

Polyps and medusae differ in regard to habitats, evolution, and morphological structures. In order to replace the former taxonomic systems which deal with polyp and medusa generations separately, by a single valid classification including both generations, knowledge of all essential life history phases is necessary. Modern methods of culturing marine hydroids are outlined briefly. Successful culturing provides the means to link formerly unidentified polyps or medusae with one another. The present paper is concerned with the hydroidEucheilota maculata Hartlaub, the medusa of which is common in the southern North Sea. The polyps formerly unknown have been reared from fertilized eggs of the medusae. They were raised to full size, formed colonies, and produced gonangia and medusae. Thus the morphology of the single polyp, the polyp colony, and of the young medusa could be investigated in detail. The nematocyst equipment of the two generations and of all developmental stages is described as well as the number of chromosomes. The systematic position of the genusEucheilota is discussed and the diagnosis of the speciesE. maculata, including the two generations, given.     

Phenotypic plasticity in hydrozoans: morph reversibility.

The life cycle of Hydrozoans typically comprises two phases: the polyp, either solitary or colonial, with generally a benthic habitat, and the medusa which lives in the plankton. In its typical metagenetic cycle, the medusa is budded from the polyp, which is the product of sexual reproduction of medusae. However, several alternative reproduction patterns have also been described. In particular some species are able to perform a regressive transformation of the medusae that transform themselves into polyps bypassing sexual reproduction. In a species with alternative morphs switched by the environment, the more labile is the correlation between environmental factors acting on the genetic switch and the factors to which the resulting form is adapted, the more hazardous will be the development of either body form. However, we can explain the evolutionary advantage offered by reversion between morphs of these plastic species living in shallow water unpredictable environments: should produced medusae be released in the "wrong" environment, they would still have a chance of survival under another form.     

Life Cycle Reversal in Aurelia sp.1 (Cnidaria,Scyphozoa)

The genus Aurelia is one of the major contributors to jellyfish blooms in coastal waters, possibly due in part to hydroclimatic and anthropogenic causes, as well as their highly adaptive reproductive traits. Despite the wide plasticity of cnidarian life cycles, especially those recognized in certain Hydroza species, the known modifications of Aurelia life history were mostly restricted to its polyp stage. In this study, we document the formation of polyps directly from the ectoderm of degenerating juvenile medusae, cell masses from medusa tissue fragments, and subumbrella of living medusae. This is the first evidence for back-transformation of sexually mature medusae into polyps in Aurelia sp.1. The resulting reconstruction of the schematic life cycle of Aurelia reveals the underestimated potential of life cycle reversal in scyphozoan medusae, with possible implications for biological and ecological studies.     

Larval necrophilia: the odd life cycle of a pandeid hydrozoan in the Weddell Sea shelf

Colonies of an athecate hydroid were found at six stations in the high Antarctic (Weddell Sea) growing on dead specimens of Flabelligera mundata (Annelida, Polychaeta). All living specimens of F. mundata at the same stations were free of epibionts. Transplantation experiments showed that hydropolyps did not produce stolons on substrates other than the epidermal jelly coat and chetae of dead F. mundata specimens. The largest colonies (>1,000 polyps) producing medusa buds were cultured until medusa liberation: growth of medusae was then surveyed for the next 5 weeks, but development of adult features was extremely slow. Young medusae were ascribed to the suborder Pandeida by the presence of two main characters, namely (1) hollow marginal tentacles, and (2) a mouth with four simple lips. Considering polyp and young medusa features, this species is acknowledged as newly recorded for the Southern Ocean, and assigned to the genus Neoturris (Hydroidomedusae, Pandeidae).     

Fish assemblage structure in urban streams of Puerto Rico: the importance of reach- and catchment-scale abiotic factors

Cotylorhiza tuberculata is a common symbiotic scyphozoan in the Mediterranean Sea. The medusae occur in extremely high abundances in enclosed coastal areas in the Mediterranean Sea. Previous laboratory experiments identified thermal control on its early life stages as the driver of medusa blooms. In the present study, new ecological aspects were tested in laboratory experiments that support the pelagic population success of this zooxanthellate jellyfish. We hypothesized that planulae larvae would have no settlement preference among substrates and that temperature would affect ephyra development, ingestion rates and daily ration. The polyp budding rate and the onset of symbiosis with zooxanthellae also were investigated. Transmission electron microscopy revealed that zooxanthella infection occurred by the polyp stage. Our results showing no substrate selectivity by planulae and high polyp budding rates in high temperatures suggest increased benthic polyp populations, which would lead to higher medusa abundances. Rates of transition from ephyrae to medusae and the feeding of early medusa stages also increased with temperature. Continuing changes in coastal ecosystems such as future climate warming and marine construction may lead to increased populations of jellyfish to the detriment of fish globally.     

Phylogeny of Medusozoa and the evolution of cnidarian life cycles

To investigate the evolution of cnidarian life cycles, data from the small subunit of the ribosome are used to derive a phylogenetic hypothesis for Medusozoa. These data indicate that Cnidaria is monophyletic and composed of Anthozoa and Medusozoa. While Cubozoa and Hydrozoa are well supported clades, Scyphozoa appears to be paraphyletic. Stauromedusae is possibly the sister group of either Cubozoa or all other medusozoans. The phylogenetic results suggest that: the polyp probably preceded the medusa in the evolution of Cnidaria; within Hydrozoa, medusa development involving the entocodon is ancestral; within Trachylina, the polyp was lost and subsequently regained in the parasitic narcomedusans; within Siphonophorae, the float originated prior to swimming bells; stauromedusans are not likely to be descended from ancestors that produced medusae by strobilation; and cubozoan polyps are simplified from those of their ancestors, which possessed polyps with gastric septa and four mesogleal muscle bands and peristomial pits.     

Species specificity in cell-substrate interactions in medusae

A new system is described for the study of ECM-tissue interactions, using the ECM (called mesogloea) of various cnidarians and isolated striated muscle and endodermal tissue of jellyfish. The mesogloea consists mainly of water and collagen. It is present in all cnidarians and can be isolated without enzyme treatment. It can be used as a substrate to which cells and tissues adhere and on which they spread and migrate. Tissues of striated muscle and endoderm adhere and spread not only on mesogloea from regions they normally cover, but also from other regions of the animal. However, adhesion and spreading are highly species-specific. Species-specific adhesion is found throughout the whole mass of mesogloea even at regions where cells do not occur naturally. The cell adhesion factor can be extracted from the mesogloea so that the mesogloea no longer shows any cell adhesion properties. The extract consists mainly of a cysteine-containing collagen.     

Activation of Rac and Cdc42 by Integrins Mediates Cell Spreading

Adhesion to ECM is required for many cell functions including cytoskeletal organization, migration, and proliferation. We observed that when cells first adhere to extracellular matrix, they spread rapidly by extending filopodia-like projections and lamellipodia. These structures are similar to the Rac- and Cdc42-dependent structures observed in growth factor-stimulated cells. We therefore investigated the involvement of Rac and Cdc42 in adhesion and spreading on the ECM protein fibronectin. We found that integrin-dependent adhesion led to the rapid activation of p21-activated kinase, a downstream effector of Cdc42 and Rac, suggesting that integrins activate at least one of these GTPases. Dominant negative mutants of Rac and Cdc42 inhibit cell spreading in such a way as to suggest that integrins activate Cdc42, which leads to the subsequent activation of Rac; both GTPases then contribute to cell spreading. These results demonstrate that initial integrin-dependent activation of Rac and Cdc42 mediates cell spreading.     

THE UPPER CELL SURFACE: ITS INABILITY TO SUPPORT ACTIVE CELL MOVEMENT IN CULTURE

A variety of epithelial cells and fibroblasts fail to move over one another's upper surfaces in culture, resulting in monolayering. The failure of seeded fibroblasts to adhere to and spread on epithelial cell surfaces suggests that monolayering in culture is due to the lack of adhesion of the upper cell surface, at least of epithelial cells. Seeded fibroblasts and postmitotic, rounded fibroblasts likewise fail to spread on the upper surfaces of spread fibroblasts, suggesting that the inability of the upper cell surface to support spreading may be a general phenomenon. Inert particles and cell processes do not adhere directly to the upper cell surface. However, they can initiate adhesions to the surface at a cell's free margin, suggesting a variation of adhesive properties over a cell's surface.     

Polypengeneration und Entwicklung vonEutonina indicans (Thecata-Leptomedusae)

The medusaEutonina indicans (Romanes 1876) represents a circumpolar northern boreal species. In the European seas it has its southern limits of distribution in the southern North Sea, where it is common during late spring and early summer. The present paper is concerned with the formerly unknown polyp generation. It was possible to rear polyps from fertilized medusa eggs to full size; they formed colonies and produced gonangia and young medusae. Thus, morphology and development of the single polyp, colony, and young medusa could be described in detail. The systematic position ofEutonina indicans is discussed briefly, and a diagnosis of the species, including the two generations, given.     

Rac Regulates Integrin-Mediated Spreading and Increased Adhesion of T Lymphocytes

Leukocyte adhesion to the extracellular matrix (ECM) is tightly controlled and is vital for the immune response. Circulating lymphocytes leave the bloodstream and adhere to ECM components at sites of inflammation and lymphoid tissues. Mechanisms for regulating T-lymphocyte–ECM adhesion include (i) an alteration in the affinity of cell surface integrin receptors for their extracellular ligands and (ii) an alteration of events following postreceptor occupancy (e.g., cell spreading). Whereas H-Ras and R-Ras were previously shown to affect T-cell adhesion by altering the affinity state of the integrin receptors, no signaling molecule has been identified for the second mechanism. In this study, we demonstrated that expression of an activated mutant of Rac triggered dramatic spreading of T cells and their increased adhesion on immobilized fibronectin in an integrin-dependent manner. This effect was not mimicked by expression of activated mutant forms of Rho, Cdc42, H-Ras, or ARF6, indicating the unique role of Rac in this event. The Rac-induced spreading was accompanied by specific cytoskeletal rearrangements. Also, a clustering of integrins at sites of cell adhesion and at the peripheral edges of spread cells was observed. We demonstrate that expression of RacV12 did not alter the level of expression of cell surface integrins or the affinity state of the integrin receptors. Moreover, our results indicate that Rac plays a role in the regulation of T-cell adhesion by a mechanism involving cell spreading, rather than by altering the level of expression or the affinity of the integrin receptors. Furthermore, we show that the Rac-mediated signaling pathway leading to spreading of T lymphocytes did not require activation of c-Jun kinase, serum response factor, or pp70^{S6 kinase} but appeared to involve a phospholipid kinase.     

Temporally and spatially restricted expression of a gland cell gene during regeneration and in vitro transdifferentiation in the hydrozoan Podocoryne carnea

An antiserum to transdifferentiated striated muscle cells from the medusa of Podocoryne carnea was prepared and used to screen a gt11-expression library prepared from gonozoids of P. carnea. We isolated a cDNA clone termed Pod-EPPT with at least 63 tandem repeats of the tetrapeptide-motive glu-pro-pro-thr, named Pod-EPPT. Using Pod-EPPT as a molecular marker for head quality the morphological relationship between the two metagenic life stages of this hydroid, the polyp and the medusa, was studied. In situ hybridization demonstrated that expression of the gene corresponding is restricted to secretory cells in the endoderm of the oral hypostome region of polyps and medusae and, presumably, to progenitor cells of this type. Cells expressing Pod-EPPT could not be observed in the larval stage. During head regeneration in polyps, Pod-EPPT expression is upregulated soon after head removal in previously non-expressing cells and in newly differentiating secretory cells. This activation of a head-specific gene precedes the morphologically obvious events of head regeneration. Pod-EPPT is one of the genes that are activated during manubrium (mouth) regeneration from experimentally combined subumbrellar plate endoderm and striated muscle of the medusa.     

Enhancement of the adhesion of fibroblasts by peptide containing an Arg-Gly-Asp sequence with poly(ethylene glycol) into a thermo-reversible hydrogel as a synthetic extracellular matrix

In an effort to regulate the behavior of mammalian cell entrapped in a gel, the gels were functionalized with the putative cell-binding (-Arg-Gly-Asp-) (RGD) domain. The adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and the cell recognition ligands were inculcated into the thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 2000) used as the biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was examined in vitro for its ability to promote cell spreading and to increase the viability of the cells by introducing PEG spacers. ECM poorly adhered to hydrogel lacking adhesion molecules permitting only a 20% spread of the seeded cells after 10 days. When the PEG spacer arms, which were immobilized by a peptide linkage, had been integrated into the hydrogel, the conjugation of RGD improved cell spreading by 600% in a 10-day trial.     

A line of rat ovarian surface epithelium provides a continuous source of complex extracellular matrix

Summary A spontaneously immortalized, yet non-tumorigenic rat ovarian surface epithelial (ROSE 199) cell line, deposits large amounts of extracellular matrix (ECM) in response to crowding. The characteristics and components of ROSE 199-derived cell-free ECM were compared after three different preparative techniques: treatment with 20 mM ammonium hydroxide, with 1% sodium deoxycholate, or by repeated freeze-thaws. The ECMs were analyzed by histochemistry, immunofluorescence, electron microscopy, and Western immunoblotting. Components of ROSE 199 ECM included laminin, fibronectin, and collagen types I and III. Even though ROSE 199 is an epithelial cell line, striated collagen fibers formed a major part of its matrix. Thus, ROSE 199 matrix consists of both basement membrane and stromal matrix components. This matrix supported the adhesion, spreading, and growth of several cell types without altering their morphology or growth pattern, and enhanced the attachment of some cell types that spread on plastic only with difficulty. Immunofluorescence, electron microscopy, and dry weight determinations indicated that a greater proportion of matrix was retained in preparations obtained by ammonium hydroxide or freeze thaw techniques than after sodium deoxycholate treatment. Ammonium hydroxide and freeze-thaw treated matrices were also superior to sodium deoxycholate preparations as evidenced by enhanced initial cellular adhesion and spreading compared to cells plated on plastic. Residual nuclear material did not seem to affect the biological activity of this matrix. ROSE 199 extracellular matrix provides a novel, complex substratum for cell culture and for studies of matrix functions and synthesis.     

Recellularization of decellularized human adipose-tissue-derived extracellular matrix sheets with other human cell types

Decellularized human extracellular matrices (ECMs) are an extremely appealing biomaterial for tissue engineering and regenerative medicine. In this study, we decellularized human adipose tissue, fabricated a thin ECM sheet and explored the potential of this human adipose-derived ECM sheet as a substrate to support the formation of tissues other than adipose tissue. Acellular ECM sheets were fabricated from human adipose tissue through successive physical and chemical treatments: homogenization, centrifugation, casting, freeze-drying and sodium dodecyl sulfate treatment. The ECM sheets exhibited good mechanical properties, despite their porous structure. They degraded quickly in the presence of collagenase and the degradation rate increased with the collagenase concentration in phosphate-buffered saline. Five different human cell types, covering a broad range of cells and applications (normal human dermal fibroblasts, human aortic smooth muscle cells, human chondrocytes, human umbilical vein endothelial cells and human adipose-derived stem cells), were seeded onto the ECM sheets. All the human cell types spread well, proliferated and were successfully integrated into the decellularized ECM sheet. Overall, the results suggest that recellularized ECM sheets are a promising substitute for defective or damaged human tissues.     

Synthesis of Arg-Gly-Asp (RGD) sequence conjugated thermo-reversible gel via the PEG spacer arm as an extracellular matrix for a pheochromocytoma cell (PC12) culture

Adhesion molecules composed of Gly-Arg-Gly-Asp-Ser (GRGDS) peptides and cell recognition ligands were inculcated into thermo-reversible hydrogel composed of N-isopropylacrylamide, with a small amount of succinyl poly(ethylene glycol) (PEG) acrylate (MW 3400) used as a biomimetic extracellular matrix (ECM). The GRGDS-containing p(NiPAAm-co-PEG) copolymer gel was studied in vitro for its ability to promote cell spreading and to increase the viability of cells by introducing PEG spacers. Hydrogel lacking the adhesion molecules proved to be a poor ECM for adhesion, permitting only a 20% spread of the seeded cells after 10 days. When PEG spacer arms, immobilized by a peptide linkage, had been integrated into the hydrogel, conjugation of RGD promoted cell spread by 600% in a 10-day trial. In addition, in a serum-free medium, only GRGDS peptides conjugated with the spacer arm were able to promote cell spread. In terms of the cell viability, GRGDS peptides conjugated with the PEG-carrying copolymer gel specifically mediated cell spread. This result supports the theory that specific recognition is the result of interaction between the integrin families on the fibroblast, and the RGD sequence on the p(NiPAAm-co-PEG) copolymer gel.     

Heat dissociation and maceration of marine Cnidaria

Summary The effect of increased temperature on the tissue integrity of polyps and medusae ofPodocoryne carnea is described. Animals exposed for 10 to 20 min to a temperature of 35°C are easily dissociated into single cells. These dissociated cells round up, form reaggregates and, depending on their origin, regenerate polyp or medusa structures. However, as the exposure time is increased, the dissociated cells gradually lose the ability to reaggregate or to regenerate defined structures. At incubation times exceeding 50 min, the tissue separates into single cells which retain their normalin vivo shapes but which do not form reaggregates. These are termed macerated cells. The ultrastructure and protein profile of macerated cells demonstrate no major changes from those of untreated cells. Both the dissociation and maceration methods are applicable to other cnidarian species for developmental, histological and biochemical studies.