Cellular and intracellular pathways mediating the metamorphic stimulus in hydrozoan planulae

Summary Both the natural metamorphic stimulus (an unidentified bacterial product) and an artificial trigger of metamorphosis (Cs⁺) cause large calcium transients in planula cells of the hydrozoanMitrocomella polydiademata. When these transients are inhibited with calcium channel blockers, metamorphosis is also inhibited. All cells of theMitrocomella planula contain a calcium-specific photoprotein. The cells where the calcium transients occur during natural- and Cs⁺-induced metamorphosis have been visualized in normal and entoderm free planulae that lack ganglion cells, using a compound microscope coupled to an image intensifier and video camera. During bacteria- and Cs⁺-induced metamorphosis, groups of contiguous cells, occupying from about 10% to the entire visible surface of the planula, simultaneously exhibit calcium transients. When the cells that initiate a transient comprise only part of the planula surface, the calcium transient frequently propagates and can eventually involve every cell on the visible planula surface. There is no special site on the planula surface where calcium transients are more apt to be initiated. There is no indication that propagation of a flash in one direction is more likely than in another. The velocity of propagation is virtually the same in all directions. The only feature of the spatial distribution of bacteria- and Cs⁺-induced calcium transients that appears to be necessary for the induction of metamorphosis is that at least one transient must involve all of the surface cells of the planula. The spatial behavior of calcium transients is the same in entoderm free planulae (lacking ganglion cells) as in normal planulae. The propagation of these calcium transients most probably occurs via epithelial conduction. This metamorphic step involving calcium transients is probably the intercellular communication system that informs the cells of the planula that metamorphosis will commence.Metamorphosis inMitrocomella planulae can also be induced with phorbol esters. Calcium transients do not occur during phorbol ester-induced metamorphosis, indicating that they act at a different point in the metamorphic pathway. Calcium channel blockers do not inhibit phorbol ester-induced metamorphosis. Inhibitors of protein kinase-C, inhibit both phorbol ester-induced metamorphosis and Cs⁺- and bacteria-induced metamorphosis, but have no effect on the calcium transients induced by Cs⁺. This indicates that the calcium transient mediated step in the metamorphic pathway occurs prior to protein kinase-C activation. Calcium transients probably play a major role in activating protein kinase-C.     

Comparative fine structural studies of planulae and primary polyps of identical age of the sea pen,Ptilosarcus gurneyl

Electron microscopic study of an 18-day-old planulae and primary polyps of the sea pen, Ptilosarcus gurneyi, reveals 14 cell types: sustentacular cell A, sustentacular cell B, nerve cell, sensory cell, cnidoblast, interstitial cell, five types of gland cell (A, B, C, D and E), amoebocyte, style cell and endodermal cell. Of these, 9 are found in the planula, 12 in polyps and 7 are common to both stages. The fine structure of all cell types is described. Since the planulae and polyps in this study were identical in age of development, the gaining and losing of certain types of cells in the polyp are attributed to changes associated with settlement and metamorphosis. Modifications of the seven common cell types during metamorphosis can also be attributed to the change of life style from pelagic to benthic.     

A fine structural study of metamorphosis of the hydrozoan Mitrocomella polydiademata

This report is a comprehensive fine structural analysis of the morphological changes occurring during metamorphosis of the marine hydrozoan Mitrocomella polydiademata. Five stages are recognized during metamorphosis: planulae just prior to settlement, ball and filiform stages, immature polyps, and primary feeding polyps. Settlement and metamorphosis of cnidarian planulae involve such changes as ciliary arrest, discharge of nematocytes, secretion of glandular cells, differentiation of cells, and changes in cell and body shape.     

Analysis of pattern formation during embryonic development of Hydractinia echinata

Summary Patterning processes during embryonic development of Hydractinia echinata were analysed for alterations in morphology and physiology as well as for changes at the cellular level by means of treatment with proportioning altering factor (PAF). PAF is an endogenous factor known to change body proportions and to stimulate nerve cell differentiation in hydroids (Plickert 1987, 1989). Applied during early embryogenesis, this factor interferes with the proper establishment of polarity in the embryo. Instead of normal shaped planulae with one single anterior and one single posterior end, larvae with multiple termini develop. Preferentially, supernumerary posterior ends, which give rise to polyp head structures during metamorphosis, form while anterior ends are reduced. The formation of such polycaudal larvae coincide with an increase in the number of interstitial cells and their derivatives at the expense of epithelial cells. Treatment of further advanced embryonic stages causes an increase in length, presumably due to the general stimulation of cell proliferation observed in such embryos. Also, the spatial arrangement of cells (i.e. cells in proliferation and RFamide (Arg-Phe-amide immunopositive nerve cells) is altered by PAF. Larvae that develop from treated embryos display altered physiological properties and are remarkably different from normal planulae with respect to their morphogenetic potential: (1) Larvae lose their capacity to regenerate missing anterior parts; isolated posterior larva fragments form regenerates of a bicaudal phenotype. (2) In accordance with the frequently observed reduction of anterior structures, the capacity to respond to metamorphosis-inducing stimuli decreases. (3) The morphogenetic potential to form basal polyp parts is found to be reduced. In contrast, the potential to form head structures during metamorphosis increases, since primary polyps with supernumerary hypostomes and tentacles metamorphose from treated animals.     

Pattern of serotonin‐like immunoreactive cells in scyphozoan and hydrozoan planulae and their relation to settlement

The planulae of almost all investigated cnidarian species possess neuron‐like cells. The distribution of these cells is usually uneven throughout the long axis of the planula. The majority of these cells are located in the anterior half of the planula body. Scyphozoan planulae, as well as anthozoan planulae, have a sensory structure at the anterior pole called an apical organ, which is believed to take part in metamorphosis induction. Hydrozoan planulae also possess sensory cells. It has been previously shown in several cnidarian larvae that their neuronal cells contain the neurotransmitter, serotonin. The present study describes the peculiarities of serotonin‐like immunoreactive cells in Aurelia aurita (Scyphozoa) and Gonothyraea loveni (Hydrozoa) planulae. We show that several cells in the presumptive apical organ of A. aurita are immunoreactive to antibodies against serotonin, while G. loveni planulae have an accumulation of serotonin‐positive cells near the anterior pole. Additional serotonin‐like immunoreactive cells are found in the lateral ectoderm of both planulae. Treatment of A. aurita and G. loveni planulae with serotonin or its blockers show that serotonin is likely involved in the initiation of planula settlement.     

Cholera toxin and thyrotropine can replace natural inducers required for the metamorphosis of larvae and buds of the scyphozoanCassiopea andromeda

Summary The scyphozoan medusaCassiopea andromeda forms free swimming planulae and buds that metamorphose into tentacle bearing sedentary polyps. About 30% of the planulae and 7% of the buds undergo such metamorphosis within 30 days in sterile natural seawater from the Red Sea. In sterile artificial sea water devoid of any organic substances, normal metamorphosis does not take place. This indicates that both the planulae and the buds require organic morphogenetic inducers present in the sea to settle and metamorphose. The addition of cholera toxin or thyrotropin to preparations of sterile artificial sea water, induced normal metamorphosis. These inducers enhanced the rate of metamorphosis and up to 100% of the planulae and buds formed polyps within 2–18 days. We conclude that our preparations of cholera toxin and thyrotropin mimic the action of natural inducers.     

Differentiation of the interstitial cell line in hydrozoan planulae

Repopulation of epithelial (colchicine-treated) planular tissue by interstitial cells, nematoblasts/nematocytes, and ganglionic cells was examined via grafting. Seventy-two-hour epithelial planular head pieces were grafted to 72-hour control labelled planular tail pieces, left in contact for 24 h, separated, and the head pieces were analyzed for interstitial cells and their derivatives. The reciprocal experiment of grafting 72-hour epithelial planular tails to 72-hour control labelled planular heads was also done and the tail pieces were examined. Repopulated planular head pieces contained interstitial cells, ganglionic cells and a reforming neural plexus but few nematoblasts/nematocytes. Reconstituted planular tail pieces contained interstitial cells and nematoblasts/nematocytes but no ganglionic cells. Results possibly suggest that the migrating interstitial cell population of 72-hour planulae is rich in committed precursors.     

Sul meccanismo di azione antimitotica della colchicina

Abstract

ON THE MECHANISM OF ACTION OF COLCHICINE ON MITOSIS. — The effect of colchicine on glutathione has been studied in order to explain its effect on mitosis. The results are consistent with the hypothesis that colchicine or its derivatives (colcemide) exert their antimitotic action by reversible oxidation of thiolic proteins present in the cell. This hypothesis could also explain the lack of antimitotic action of thiocol-chisode.     

Role of the cellular environment in interstitial stem cell proliferation in Hydra

Summary The role of the cellular environment on hydra stem cell proliferation and differentiation was investigated by introduction of interstitial cells into host tissue of defined cellular composition. In epithelial tissue lacking all non-epithelial cells the interstitial cell population did not grow but differentiated into nerve cells and nematocytes. In host tissue with progressively increased numbers of nerve cells growth of the interstitial cell population was positively correlated to the nerve cell density. In agreement with previous observations (Bode et al. 1976), growth of the interstitial cell population was also found to be negatively correlated to the level of interstitial cells present. The strong correlation between the growth of the interstitial cell population and the presence of interstitial cells and nerve cells implies that interstitial cell proliferation is controlled by a feedback signal from interstitial cells and their derivatives. Our results suggest that the cellular environment of interstitial cells provides cues which are instrumental in stem cell decision making. Offprint requests to: T.C.G. Bosch     

Metamorphosis and acquisition of symbiotic algae in planula larvae and primary polyps of Acropora spp.

Coral planulae settle, then metamorphose and form polyps. This study examined the morphological process of metamorphosis from planulae into primary polyps in the scleractinian corals Acropora nobilis and Acropora microphthalma, using the cnidarian neuropeptide Hym-248. These two species release eggs that do not contain Symbiodinium. The mode of acquisition of freshly isolated Symbiodinium (zooxanthellae) (FIZ) by the non-symbiotic polyp was also examined. Non-Hym-248 treated swimming Acropora planulae did not develop blastopore, mesenteries or coelenteron until the induction of metamorphosis 16 days after fertilization. The oral pore was formed by invagination of the epidermal layer after formation of the coelenteron in metamorphosing polyps. At 3 days after settlement and metamorphosis, primary polyps exposed to FIZ established symbioses with the Symbiodinium. Two–four days after exposure to FIZ, the distribution of Symbiodinium was limited to the gastrodermis of the pharynx and basal part of the polyps. Eight–ten days after exposure to FIZ, Symbiodinium were present in gastrodermal cells throughout the polyps.     

SOMATOSTATIN-LIKE IMMUNOREACTIVE CELLS IN THE GROUND SQUIRREL RETINA

Immunocytochemical techniques were employed to locate somatostatin (SS)-containing cells in the retina of the 13-lined ground squirrel (Spermophilus tridecemlineatus). In normal retinas immunostain was limited to neuronal processes, yet distinctly labeled somata were detected in retinas of animals pretreated with colchicine. Labeled cell bodies were located in the outermost and innermost portions of the inner nuclear layer (INL) and in the ganglion cell layer (GCL). The largest population of SS-like immunoreactive neurons was found in the innermost INL. These cells were identified as small and medium sized amacrine cells whose soma diameters ranged from 4 to 14μm. A smaller population of immunoreactive cells was observed in the outermost region of the INL. These cells, presumptive horizontal cells, were found mainly in peripheral regions of the retina. Immunoreactive cells in the GCL were of two types: displaced amacrines, and retinal ganglion cells. SS-positive axons in the optic fiber layer suggest that some of the immunoreactive GCL neurons were ganglion cells, and it is our opinion that these cells belong to a class of associational ganglion cells previously identified in other species.     

Genital primordium of the free-living marine nematode Halichoanolaimus sonorus (Chromadoria,Chromadorida)

Summary

The genital primordium of the first stage juvenile (J1) of the free-living marine nematode Halichoanolaimus sonorus (Chromadorida: Selachinematidae) was studied using transmission electron microscopy. The primordium consists of four undifferentiated cells: two primordial germ cells (PGC) 5–6 μm in diameter and two somatic cells. The PGC have a large nucleus with nucleolus. The centriole was detected in close vicinity of the PGC nucleus. Most of the cell mitochondria are in close contact with the nuclear envelope. The mitochondria are interspersed by 0.2–0.3 μm particles of an electron-dense diffuse substance devoid of surrounding membrane. Both PGC are closely attached to each other and to the neighboring somatic cells of the genital primordium. The elongated somatic cells contain nuclei devoid of nucleoli; the cytoplasm is filled with free ribosomes and contains occasional cisternae of rough endoplasmatic reticulum (RER), Golgi bodies, mitochondria, and transparent vesicles. The genital primordium is separated by a narrow space from of the intestine (dorsally) and the somatic muscles (ventrally). The PGC of H. sonorous are devoid of typical P granules known for previously studied nematodes as distinct markers of germ line cell lineage. Perinuclear particles of dense diffuse substance found in PGC of H. sonorous could be considered as germ determinants analogous to P granules.     

Effects of cycloSal-D4TMP Derivatives in H9 Cells with Induced AZT Resistance Phenotype

Abstract

Cytotoxic and antiretroviral activity of cycloSal-d4TMP derivatives were tested in a new AZT-resistant H9 cell subline (H9^rAZT²⁵⁰). The results showed, that cycloSal-d4TMP derivatives overcame resistance of HIV-1 to d4T in H9^rAZT²⁵⁰ cells, which exert decreased thymidine kinase (TK) gene expression.     

Herpes Simplex Virus Thymidine Kinase Gene-Transfected Tumor Cells; Sensitivity to Antiherpetic Drugs

Abstract

A series of antiherpetic 5-substituted 2′-deoxyuridine derivatives (i. e. BVDU) and guanine derivatives (i. e. ganciclovir) have been evaluated for their cytostatic activity against murine mammary carcinoma FM3A cell lines that are deficient in cytosol thymidine kinase, but transfected by the herpes simplex virus type 1 (HSV-1)- or type 2 (HSV-2)-specified thymidine kinase gene. Most compounds were endowed with a markedly higher cytostatic activity against the HSV TK gene-transfected tumor cells than against wild-type tumor cells. The principal target for cytostatic activity of the BVDU derivatives proved thymidylate synthase, whereas the guanine derivatives inhibited HSV TK gene-transfected tumor cell proliferation by competing with cellular DNA polymerase(s) and subsequent incorporation into the cellular genome.

     

Implication of Catecholamines During Spawning in Marine Bivalve Molluscs

Summary

During the tail-bud stage of Ascidiella aspersa embryogenesis, the test cells or innermost cells of the egg envelope manifest locomotive activities. Light microscopy further reveals that the adhesive behaviour of test cells changes in the course of embryogenesis. Mechanical dechorionation experiments performed on 846 embryos demonstrate that up to the tail-bud stage all test cells are attached to the inner surface of the chorion. The embryo is completely devoid of test cells. At the onset of larval tunic secretion, increasing numbers of test cells settle on the embryo until all test cells adhere to it. This switch in adhesive properties is completed within 65 min. The hatched larva carries the entire complement of test cells until the onset of metamorphosis. SEM and observations show that test cells do not establish direct cell-to-cell contacts to ectodermal cells but attach to the larval tunic.     

Appearance of Dendritic Cells in the Basal Layer of Corneal Epithelium during Metamorphosis in Rana temporaria L.

A population of dendritic cells was found at the light optic level in the basal layer of the corneal epithelium inRana temporaria L. The cells had wide short processes which penetrated between neighboring epithelial cells. The cell bases lied on the basal membrane while the bodies are slightly elongated perpendicularly to the epithelium surface. The appearance of dendritic cells coincided in time with the beginning of metamorphosis of the tadpoles and their number increased by the end of metamorphosis. The dendritic cells we described were similar in their morphology to the Langerhans cells.     

Changes in Adhesion of Candida tropicalis Clinical Isolates Exhibiting Switch Phenotypes to Polystyrene and HeLa Cells

Background

Candida tropicalis is an important human pathogen that can undergo multiple forms of phenotypic switching.

Aim

We aimed to evaluate the effect of phenotypic switching on the adhesion ability of C. tropicalis.

Methods

C. tropicalis morphotypes included parental phenotypes (clinical isolates) and switch phenotypes (crepe, revertant of crepe—CR, rough, revertant of rough—RR, irregular center and revertant of irregular center—ICR). Adhesion to polystyrene and HeLa cells was determined by crystal violet assay. The percentage of HeLa cells with adhered yeasts and the number of adhered yeasts per HeLa cell were determined by light microscopy. Filamentation among adhered cells was assessed by direct counting.

Results

On polystyrene, 60% of the switch strains showed difference (p?<?0.05) on adhesion ability compared to their parental counterpart strains, and altered thickness of adhered cells layers. Filamentation was increased among adhered cells of the switched strains compared to parental strains. A positive correlation was observed between adhesion on polystyrene and filamentation for morphotypes of the system 49.07. The majority of the switched strains showed higher adhesion capability to HeLa cells in comparison to the adherence of the clinical strains. All revertant strains showed a higher number of yeast cells per HeLa cell compared to their variant counterparts (p?<?0.05), with exception of the ICR.

Conclusions

Our findings indicate that switching events in C. tropicalis affect adhesion and filamentation of adhered cells on polystyrene and HeLa cells. The rise of switch strains with increased adhesion ability may contribute to the success of infection associated with C. tropicalis.

     

Neuroprotective effect of Korea Red Ginseng extract on 1-methyl-4-phenylpyridinium-induced apoptosis in PC12 Cells

Background: Korean Red Ginseng (KRG) is a valuable herb in Asian countries that is used as a crude substance to inhibit inflammation and to enhance vitality, longevity and immunity. The protective effects of KRG against the toxicity of 1-methyl-4-phenylpyridinium (MPP+) were investigated in vitro in the present study.

Methods: PC12 cells were pretreated with the water extract of KRG for 24?h, then incubated with MPP+ for 24?h. The growth of the cells was assessed using a live cell viability assay, the ratio of apoptotic cells was measured using flow cytometry and morphology of the apoptotic cells was detected using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. The expressions of caspase-3 and -9 were measured by quantitative real-time PCR.

Results: Pretreatment of the KRG extract increased cell viability significantly when compared with that of only MPP+-treated cells (p?p?p?p?Conclusion: These results indicate that KRG treatment suppresses MPP+-induced apoptosis in PC12 cells by regulating caspase cascades, suggesting a possible role for KRG in the prevention and treatment of Parkinson’s disease.     

Localization of sensory mechanisms utilized by coral planulae to detect settlement cues

Coral planulae are induced to settle and metamorphose by contact with either crustose coralline algae or marine bacterial biofilms. Larvae of two coral species, Pocillopora damicornis and Montipora capitata, which respond to different metamorphic cues, were utilized to investigate the sensory mechanisms used to detect metamorphic cues. Because the aboral pole of the coral planula is the point of attachment to the substratum, we predicted that it is also the point of detection for cues. To determine where sensory cells for cues are localized along the body, individual larvae were transversely cut into oral and aboral portions at various levels along the oral–aboral axis, and exposed to settlement‐inducing substrata. Aboral ends of M. capitata metamorphosed, while oral ends continued to swim. However, in larvae of P. damicornis, ¾ oral ends (i.e., lacking the aboral pole) were also able to metamorphose, indicating that the cells that detect cues may be distributed along the sides of the body. These cells do not correspond to FMRFamide‐immunoreactive cells that are present throughout the body. Cesium ions induced both aboral and oral ends of larvae of both species to settle, suggesting that oral ends have not lost their capacity to metamorphose, despite lacking sensory cells to detect natural cues. To determine whether sensory cells in larvae of P. damicornis are restricted to one side of the body, swimming behavior over substrata was observed in larvae labeled with diI, a red fluorescent lipophilic membrane stain. The larvae were found to rotate around the oral–aboral axis, with their surface against the substratum, not favoring a particular side for detecting cues. While clarifying the regions of the larval body important for settlement and metamorphosis in coral planulae, we conclude that significant differences between coral species may be due to differences in the distribution of sensory structures in relation to different planular sizes.     

Metamorphosis of <Emphasis Type="Italic">Hydractinia echinata</Emphasis>—natural versus artificial induction and developmental plasticity

Many marine invertebrates reproduce through a larval stage. The settlement and metamorphosis of most of the species are synchronised and induced by environmental organisms, mainly bacteria. The hydrozoan Hydractinia echinata has become a model organism for metamorphosis of marine invertebrates. In this species, bacteria, e.g. Pseudoalteromonas espejiana, are the natural inducers of metamorphosis. Like in other species of marine invertebrates, metamorphosis can be induced artificially by monovalent cations, e.g. Cs⁺. In this study, we present systematic data that metamorphosis—with both inducing compounds, the natural one from bacteria and the artificial one Cs⁺—are indeed similar with respect to (a) the morphological progression, (b) the localisation of the primary induction signal in the larva, (c) the pattern of apoptotic cells occurring during the initial 10 h of metamorphosis and (d) the disappearance of RFamide-dependent immunocytochemical signals in sensory neurons during this process. However, a difference occurs during the development of the anterior end, insofar as apoptotic cells and settlement appear earlier in planulae induced with bacteria. Thus, basically, Cs⁺ may be used as an artificial inducer, mimicking the natural process. However, differences in the appearance of apoptotic cells and in settlement raise the question of how enormous developmental plasticity in hydrozoans actually can be, and how this is related to the absence of malignant devolution in hydrozoans.