Articulating the "stem cell niche" paradigm through the lens of non-model aquatic invertebrates.

Stem cells (SCs) in vertebrates typically reside in "stem cell niches" (SCNs), morphologically restricted tissue microenvironments that are important for SC survival and proliferation. SCNs are broadly defined by properties including physical location, but in contrast to vertebrates and other "model" organisms, aquatic invertebrate SCs do not have clearly documented niche outlines or properties. Life strategies such as regeneration or asexual reproduction may have conditioned the niche architectural variability in aquatic or marine animal groups. By both establishing the invertebrates SCNs as independent types, yet allowing inclusiveness among them, the comparative analysis will allow the future functional characterization of SCNs.

Molecular characterization and metal induced gene expression of the novel glutathione peroxidase 7 from the chordate invertebrate Ciona robusta.

In the present study, we describe the identified and characterized the gene and the transcript of a novel glutathione peroxidase-7 (GPx7) from the solitary ascidian Ciona robusta, an invertebrate chordate widely distributed in temperate shallow seawater. The putative nucleotide and amino acid sequences were compared with those of GPx7 from other metazoans and phylogenetic analysis suggests the presence of a high evolutionary pressure in the contest of neutral evolution. The mRNA of CrGPx7 is located in hemocytes and ovarian follicular cells, as revealed by in situ hybridization. The time course of CrGPx7 mRNA levels in the presence of Cd, Cu and Zn, showed upregulation in the final stages of the experiments, suggesting a role of GPx7 in late protection from oxidative stress. Our in silico analyses of the crgpx7 promoter region revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and xenobiotic-responsive elements (XRE), suggesting that the transcription of these genes directly depends on metals. Cell-free extract from C. robusta tissues show the presence of selenium-independent GPx activity that is inhibited by the presence of metals.

Transcription of genes involved in glutathione biosynthesis in the solitary tunicate Ciona intestinalis exposed to metals.

Exposure to metals is known to generate oxidative stress risk in living organisms, which are able to respond with the induction of antioxidant defenses, both enzymatic and non-enzymatic. Glutathione (GSH) is considered to be an important cellular component involved in protecting cells, both as metal chelating agent and oxygen radical scavenger. In this work we used molecular techniques to analyze the nucleotide and predicted amino acid sequences of genes involved in GSH biosynthesis, γ-glutamyl-cysteine ligase catalytic subunit (ci-gclc), γ-glutamyl-cysteine ligase modifier subunit (ci-gclm) and GSH synthase (ci-gs) in the solitary tunicate Ciona intestinalis. We also studied the transcription of the above genes after in vivo exposure to Cd, Cu and Zn by semiquantitativ RT-PCR to improve our knowledge about the relationship between metal-induced oxidative stress and GSH production and locate mRNA expression by in situ hybridization (ISH). These genes exhibit a good level of sequence conservation with metazoan homologs generally, especially for residues important for the activity of the enzymes. Phylogenetic analyses indicate that the three enzymes evolved in different ways, Ci-GCLC and Ci-GS being mostly correlated with invertebrate proteins, Ci-GCLM being as sister group of vertebrate GCLMs. Our in silico analyses of the ci-gs and ci-gclc promoter regions revealed putative consensus sequences similar to mammalian metal-responsive elements (MRE) and antioxidant response elements (ARE), indicating that the transcription of these genes may directly depend on metals and/or reactive oxygen species. Results highlight a statistically significant increase in gene transcription, demonstrating that metal treatments have inducible effects on these genes. They can modulate gene transcription not only through MREs but also through AREs, as a consequence of metal-dependent ROS formation. The ISH location of Ci-GS and Ci-GCLC mRNAs shows that the cells most involved in glutathione biosynthesis are circulating hemocytes. The data presented here emphasize the importance of complex metal regulation of ci-gclc, ci-gclm and ci-gs transcription, which can create an efficient detoxification pathway allowing C. intestinalis to survive in continued elevated presence of metals in the environment.

Botryllus schlosseri: a model ascidian for the study of asexual reproduction.

Botryllus schlosseri, a cosmopolitan colonial ascidian reared in the laboratory for more than 50 years, reproduces both sexually and asexually and is used as a model organism for studying a variety of biological problems. Colonies are formed of numerous, genetically identical individuals (zooids) and undergo cyclical generation changes in which the adult zooids die and are replaced by their maturing buds. Because the progression of the colonial life cycle is intimately correlated with blastogenesis, a shared staging method of bud development is required to compare data coming from different laboratories. With the present review, we aim (1) to introduce B. schlosseri as a valuable chordate model to study various biological problems and, especially, sexual and asexual development; (2) to offer a detailed description of bud development up to adulthood and the attainment of sexual maturity; (3) to re-examine Sabbadin's (1955) staging method and re-propose it as a simple tool for in vivo recognition of the main morphogenetic events and recurrent changes in the blastogenetic cycle, as it refers to the developmental stages of buds and adults.

Programmed cell death in vegetative development: apoptosis during the colonial life cycle of the ascidian Botryllus schlosseri.

Programmed cell death (PCD) by apoptosis is a physiological mechanism by which cells are eliminated during embryonic and post-embryonic stages of animal life cycle. During asexual reproduction, the zooids of colonial ascidians originate from an assorted cell population instead of a single zygote, so that we assume that regulation of the equilibrium among proliferation, differentiation and cell death may follow different pathways in comparison to the embryonic development. Here we investigate the presence of apoptotic events throughout the blastogenetic life cycle of the colonial ascidian Botryllus schlosseri, by means of terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) coupled with histochemical and electron microscopy techniques. The occurrence of low levels of morphogenetic cell death suggests that, in contrast to what happens during sexual development (embryogenesis and metamorphosis), apoptosis does not play a pivotal role during asexual propagation in botryllid ascidian. Nevertheless, PCD emerges as a key force to regulate homeostasis in adult zooids and to shape and modulate the growth of the whole colony.

Cytochemical properties of Botryllus schlosseri haemocytes: indications for morpho-functional characterisation.

In the present study, we carried out a detailed light microscopy investigation of the cytochemical properties of the haemocytes of the colonial ascidian Botryllus schlosseri, using new cytochemical stains and enzymatic markers, a panel of antibodies and lectins as probes to characterise Botryllus blood cells further. Results indicate that lymphocyte-like cells are circulating undifferentiated cells recognised by anti-CD34 antibody and there are at least two defined haemocyte differentiation pathways: i) phagocytes, represented by hyaline amoebocytes and macrophage-like cells, which share similar staining properties, the same hydrolytic enzyme content as well as the presence of detectable cytochrome-c-oxidase activity, recognition by anti-CD39 and Narcissus pseudonarcissus agglutinin; ii) cytotoxic cell line, represented by granular amoebocytes and morula cells which have vacuoles stained by Ehrlich's stain and Neutral Red; DOPA-containing protein are present inside morula cell vacuoles. Pigment cells and nephrocytes are involved in catabolite storage but their relationships with other cell types are less clear.

The spherule cells of Holothuria polii Delle Chiaie, 1823 (Aspidochirota, Holothuroidea) during brown body formation: an ultrastructural study.

Spherule cells are specific types of coelomocytes found in both the coelomic fluids and the connective tissues of many echinoderm groups and are characterised by large membrane-bound inclusions which completely fill their cytoplasm. In holothurians they are present in massive number in the coelomic fluids and are employed in brown body formation. Brown bodies are products of encapsulation and mainly consist of phagocytic amoebocytes and spherule cells: they surround foreign particles too large to be ingested by circulating phagocytes. During brown body formation, phagocytic amoebocytes flatten out over the surface of foreign particles to form unpigmented nodules which eventually aggregate into a single brown body in which many spherule cells are entrapped. Morphological modifications of spherule cells were studied in Holothuria polii following the induction of brown body formation by intracoelomic injection of sheep erythrocytes. Our ultrastructural observations provide evidence that the granules undergo typical exocytosis after previous disorganisation of their content and suggest a specific secretory activity for the spherule cells. The possible functional role of the secreted vacuolar material in brown body formation is discussed.

Morula cells as the major immunomodulatory hemocytes in ascidians: evidences from the colonial species Botryllus schlosseri.

Immunocytochemical methods were used to study the presence and distribution of IL-1-alpha- and TNF-alpha-like molecules in the hemocytes of the colonial ascidian Botryllus schlosseri. Only a few unstimulated hemocytes were positive to both the antibodies used. When the hemocytes were stimulated with either mannan or phorbol 12-mono-myristate, the phagocytes were not significantly changed in their number, staining intensity, or cell morphology. In contrast, stimulated morula cells were intensely labeled, indicating that these cells play an important immunomodulatory role.

Effects of copper and cadmium exposure on functional responses of hemocytes in the clam, Tapes philippinarum.

The effects of Cu and Cd on functional responses of hemocytes in Tapes philippinarum, a Veneridae clam widespread in the lagoon of Venice, were evaluated. Bivalves were exposed for 7 days to various concentrations of CuCl(2) x 2H(2)O (0, 10, 60, and 110 microg/L) and CdCl(2) x H(2)O (0, 150, 300, and 450 microg/L). Hemocytes were collected from exposed clams, and the effects of Cu and Cd on phagocytosis, Neutral Red retention capacity, superoxide dismutase, and cytochrome oxidase activities were investigated. Hemocytes from animals exposed to Cu showed a significant decrease in phagocytic activity, whereas no inhibition was observed in cells from Cd-exposed animals. Diffusion of Neutral Red into the cytosol, an index of lysosomal membrane alteration, was enhanced by high concentrations of both metals. Different results in biochemical parameters were observed. Exposure to 60 and 110 microg Cu/L caused a significant reduction in hemocyte superoxide dismutase activity, whereas no changes resulted after Cd exposure. Increased cytochrome oxidase activity was observed in hemocytes from mollusks exposed to 60 microg Cu/L and 300 microg Cd/L; no significant differences were found in cells from bivalves exposed to 110 microg Cu/L and 450 microg Cd/L. These results suggest a relationship between heavy metal exposure and alterations in functional responses of hemocytes in T. philippinarum and indicate that the type of observed effects vary with the nature and concentration of heavy metals. Moreover, the data obtained in the analyzed clam support the possibility of using it as sentinel organism in biomonitoring studies, even if used biomarkers will be further evaluated in field conditions.

Tributyltin induces cytoskeletal alterations in the colonial ascidian Botryllus schlosseri phagocytes via interaction with calmodulin.

In the colonial ascidian Botryllus schlosseri, tributyltin (TBT), a powerful antifouling biocide, acts as immunotoxic xenobiotic since, at a sublethal concentration (10 µM), it causes an irreversible and significant decrease in in vitro yeast phagocytosis, associated with considerable changes in the shape of phagocytes, which withdraw their pseudopodia and become spherical, due to structural damage of cytoskeletal components. The addition of TBT to the culture medium causes a significant decrease in the amoebocytic index, i.e. the percentage of amoeboid-shaped haemocytes, and prolonged washing in sea water never succeeds in restoring amoeboid shape. In these cytoskeletal alterations, F-actin undergoes extensive depolymerisation, resulting in the absence of FITC-phalloidin fluorescence. Microtubules are not recognisable as single filaments with anti-alpha-tubulin immunofluorescence, although the centrosome is not affected. The addition of increasing exogenous calmodulin (CaM) concentrations (from 20 to 120 µM) after incubation in TBT determines a significant increase in the amoebocytic index, although it is not able to bring it to that of controls, suggesting that CaM in the medium in any case externally exerts an influence on haemocytes pretreated with TBT. The copresence of TBT and exogenous CaM at concentrations higher than 80 µg/ml restores the amoebocytic index and cytoskeletal morphology. The latter appears complete for microtubules and partial for microfilaments. Experiments with isodynamic mixtures of TBT and specific CaM inhibitors, i.e. chlorpromazine (CPZ) and N-(6-aminohexyl)-5-chloronaphtalene-1-sulfonamide (W-7), reveal the synergistic effect of antagonism, indicating competition for the same site - a Ca(2+)-CaM hydrophobic region - by both interacting substances and, therefore, the formation of a TBT-CaM complex. Instead, isodynamic mixtures with thapsigargin, an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, have an effect of potentiation, suggesting that TBT indirectly interacts with this Ca(2+)-ATPase activity. We hypothesise that the main mechanism of action of TBT in B. schlosseri phagocytes is alteration of Ca(2+) homeostasis by means of direct interaction with endogenous CaM, which induces a conformational change preventing the regulative activity of CaM on Ca(2+)-ATPase. Consequently, an excess of cytosolic Ca(2+) accumulates which, together with the inhibition of CaM-dependent kinases and Ca(2+)-regulated proteins, produces extensive cytoskeletal disorganisation.

Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation.

Tapes philippinarum is a bivalve mollusc of the Pacific Ocean, successfully imported for human consumption into the northern Adriatic Sea (Europe). For better knowledge of its considerable adaptive ability in comparison with similar autochthonous species, a morpho-functional characterisation of its haemocytes was carried out with the establishment of short-term cell cultures (60 min at 25 degrees C). Various methods of cytochemical staining identified four cell types in the haemolymph: granulocytes (48.05% +/- 1.43), hyalinocytes (32.18% +/- 0.99), haemoblasts (18.97% +/- 0.63) and serous cells (0.8% +/- 0.19). The granulocytes, possessing cytoplasmic granules with differing dye affinity, included basophils, neutrophils and acidophils. Such granules stained vitally with Neutral Red, and correspond to lysosomes. Hydrolytic and oxidative enzymes were mainly detectable after stimulation in the presence of yeast cells. Both granulocytes and hyalinocytes were positive for alkaline phosphatase, non-specific esterase, peroxidase, and cytochrome C oxidase, whereas only granulocytes were positive for beta-glucuronidase, acid esterase, and arylsulphatase. Both cell types were competent phagocytes towards yeast and plasma had an opsonising effect. Moreover, the respiratory burst accompanied phagocytosis with superoxide anion production, recognisable through cytoplasmic deposits of formazan after treatment with nitro blue tetrazolium. Haemoblasts were small undifferentiated cells which, due to their morphology and positivity to the anti-CD34 antibody, show the typical features of stem cells. Serous cells, probably arising from Keber's gland and belonging to another differentiation pathway, contained non-sulphate acid mucopolysaccharides and play an important role in early defence mechanisms, taking part in the formation of clots.

Purification and characterization of a humoral opsonin, with specificity for D-galactose, in the colonial ascidian Botryllus schlosseri.

A humoral agglutinin from the hemolysate of the colonial ascidian Botryllus schlosseri was purified by affinity chromatography. This agglutinin does not require metal cations for its activity and is specific for derivatives of D-galactose. On SDS-PAGE analysis, it was resolved in two bands, of 17 and 19 kDa in reducing conditions and 15 and 16 kDa in non-reducing conditions. This behavior is due to the establishment of disulfide bridge between the thiols of cysteine, well represented in the molecule as revealed by amino acid analysis. The latter also indicated high percentages of hydrophilic residues, probably involved in sugar recognition. The lectin is an opsonin, as it increases both the phagocytic index and the number of phagocytized yeast cells. The hypothesis that this Botryllus agglutinin belongs to the galectin family of lectins is discussed.

A unique Na+/H+ exchanger, analogous to NHE1, in the chicken embryonic fibroblast.

We report the characterization of an Na+/H+ exchanger (NHE) in embryonic fibroblasts (SL-29 cells) of the chicken, a terrestrial vertebrate, where Na+ conservation is important. This exchanger is electroneutral, has a single Na+ binding site, and is highly sensitive to amiloride (IC50 2 microM), dimethyl amiloride (350 nM), and ethyl-isopropyl amiloride (25 nM). It is stimulated by serum, transforming growth factor-alpha, hypertonicity, and okadaic acid. Although these features make it resemble mammalian NHE1, other characteristics suggest distinct differences. First, in contrast to mammalian NHE1 it is inhibited by cAMP and shows a biphasic response to phorbol esters and a highly variable response to increased intracellular Ca2+ concentration. Second, whereas full-length human and rat NHE1 cDNA probes recognize a 4.8-kb transcript in rat tissues, they recognize only a 3.9-kb transcript in chicken tissues. An antibody against amino acids 631-746 of human NHE1 sequence fails to recognize a protein in SL-29 cells. Rat NHE2 and NHE3 probes do not recognize any transcript in chicken fibroblasts. The SL-29 exchanger differs markedly from the previously characterized chicken intestinal apical exchanger in its amiloride sensitivity and regulation by phorbol esters. These results suggest that a modified version of mammalian NHE1 is present in chicken tissues and imply that another functionally distinct Na+/H+ exchanger is expressed in aves.

Immunotoxic effects of organotin compounds in Tapes philippinarum.

One of the most harmful groups of coastal pollutants is the organotin compounds (OTCs) which have severe effects on both aquatic organisms and mammals including humans. The immunotoxic effects of OTCs were studied in the cultivated clam Tapes philippinarum by determining the immunosuppressant role on in vitro yeast phagocytosis at low doses (0.01, 0.05, 0.1 microM). The phagocytic index was significantly reduced in an irreversible non-lethal manner depending on concentration and lipophilic affinity. The order of inhibition was TBT > or = DBT > MBT for butyltins and TPTC > TPTA > or = TPTH for triphenyltins.

Cytoskeleton alterations by tributyltin (TBT) in tunicate phagocytes.

The effects of tributyltin chloride (TBT) on cytoskeletal components, as possible cell targets of toxicity, were examined on cultured hemocytes of the colonial ascidian Botryllus schlosseri by means of indirect immunofluorescence. The immunotoxic effect of 10 microM TBT (sublethal concentration) consists of (1) inhibition of yeast phagocytosis, Ca2+ ATPase activity, and respiratory burst; (2) increase in intracellular Ca2+ concentration; and (3) alterations in cell morphology. After 60 min, TBT-exposed amebocytes become spherical, withdrawing their long pseudopodia, and lose motility. Their microfilaments assemble in clusters around the peripheric cytoplasm, indicating massive disassembly, with the exception of unaltered adhesion plaques. Analogously, their microtubules reveal extensive disaggregation, being scattered in the cytoplasm and not recognizable as single filaments, whereas the microtubule organizing center (MTOC) is still visible. Treatment together with 20 micrograms/ml calmodulin (CaM) can partially restore the cytoskeleton architecture. These results suggest a relationship between TBT and Ca2+ homeostasis in ascidian hemocytes. By interfering with Ca2+ ATPase activity through CaM inhibition, either directly or indirectly, TBT induces an excess of intracellular Ca2+ accumulation, which first causes internal disorganization of cytoskeletal proteins and consequently inhibition of phagocytosis, beginning from chemotaxis and particle adhesion.

Phenoloxidase and cytotoxicity in the compound ascidian Botryllus schlosseri.

The vacuoles of morula cells (MC) of the colonial ascidian Botryllus schlosseri contain phenoloxidase (PO). As the release of their vacuolar content at the border of incompatible contacting colonies is associated with the formation of necrotic masses which characterize the rejection reaction, the role of PO in Botryllus cytotoxicity was investigated. When hemocytes are incubated with blood plasma from incompatible (heterologous) colonies, MC degranulate and, after 60 min, the cytotoxicity index becomes significantly greater than that observed in controls incubated with autologous plasma. The rise in cell mortality is completely inhibited by the addition of PO inhibitors sodium benzoate, tropolone and phenylthiourea, and serine protease inhibitors phenylmethylsulfonyl fluoride, benzamidine, N-tosyl-L-phenylalanine chloromethyl ketone and N-tosyl-L-lysine chloromethyl ketone. The addition of either reducing agents L-cysteine and ascorbic acid or reactive oxygen species scavenger enzymes superoxide dismutase and catalase has a similar effect. Significant inhibition of cytotoxicity is also observed with the quinone scavenger, 3-methyl-2-benzothiazolinone hydrazone. In the presence of sodium benzoate and phenylthiourea, there is a significant reduction in the number, size and color intensity of necrotic masses along the contact border of incompatible colonies. A significant increase in superoxide anion production, completely inhibited by sodium benzoate, is observed when hemocytes are incubated with heterologous blood plasma. These results indicate that: (i) PO is the enzyme responsible for the cytotoxicity observed in both hemocyte cultures and rejection reactions; (ii) PO is present inside MC vacuoles as a proenzyme which is activated, upon release, by humoral proteases; (iii) cytotoxicity appears to be mainly due to oxidative stress generated by PO during oxidation of polyphenols to quinones without the involvement of other oxidases such as NADPH oxidase and peroxidase.

Calcium homeostasis and yeast phagocytosis in hemocytes of the colonial ascidian Botryllus schlosseri.

This paper reports the effects of drugs affecting the homeostasis of cytosolic-free calcium on in vitro yeast phagocytosis by hemocytes of the colonial ascidian Botryllus schlosseri. Significant inhibition of phagocytosis is observed after exposure of hemocytes to 10 microM or higher concentrations of thimerosal, which is known to deplete intracellular calcium stores in mammalian cells. The two calcium channel blockers nifedipine and verapamil significantly decrease the phagocytic index, the minimum effective concentrations being 10 and 50 microM, respectively. As these substances have no effects at lower concentrations, they probably act through the inhibition of Ca(2+)-ATPase activity, required to restock intracellular calcium stores, due to their interaction with calmodulin. Analogously, pimozide, which suppresses ATPase activity by interacting with calmodulin, and thapsigargin, which inhibits Ca(2+)-ATPase activity, significantly reduce the phagocytic index. Moreover, nifedipine, by altering cytosolic calcium homeostasis, also lowers the production of superoxide anion associated with phagocytosis. Results indicate that in ascidians, as in mammals, a rise in intracellular calcium is required for phagocyte activation and induction of the respiratory burst.

Toxicity of organotin compounds on embryos of a marine invertebrate (Styela plicata; tunicata).

In order to clarify the interaction mechanism between organotin compounds and organisms, the effects of these compounds on the development of a benthonic filter-feeding invertebrate were studied. Embryos of the ascidian Styela plicata were obtained in laboratory by cross-fertilization and their development was followed in vivo after incubation with 0.1, 1, and 10 microM organotin compounds for various exposure times. Moreover, embryos selected at opportune stages after incubation with 10 microM tributyltin (TBT) or triphenyltin (TPT) for 1 hr were observed at the electron microscope to recognize cell alterations. Results indicate that organotins significantly affect all stages of ascidian development in a dose- and time-dependent manner and the most sensitive stages are gastrula and neurula. These compounds are able to block development, giving rise to anomalous embryos with irreversible effects. The order of inhibition appears to be strongly dependent on the organotin liposolubility: TBT > dibutyltin (DBT) > monobutyltin (MBT) and TPT > tricyclohexyltin (TCHT). The mitosis block of blastomeres in the early stages may be related to an inhibition of the microtubule polymerization. Observations with light and electron microscopes reveal globeshaped blastomeres with large intercellular spaces in the morula and gastrula stages, suggesting a toxic damage with alteration of the cytoskeleton. Moreover, the occurrence of electron-dense precipitates of organotins in the inner membrane of mitochondria and morphological changes of their cristae suggest an inhibitory effect on oxidative phosphorylation which is conspicuous in the gastrula stage. In this stage, the size of the electron-dense aggregates grow from 50-70 to 110-170 nm, while at the same time the alteration of the cristae increases.

Phagocytosis in the colonial ascidian Botryllus schlosseri.

Phagocytosis by Botryllus schlosseri hemocytes is influenced by temperature, pH, concentration, and physicochemical properties of the test particles and requires Ca2+ or Mg2+ ions to occur. Phagocytes recognize glucosyl or mannosyl residues on the surface of yeast cells, and a respiratory burst is associated with phagocytosis, as indicated by increased superoxide production. Factors that enhance phagocytosis of yeast, sheep red blood cells, and latex beads and reduce the uptake of yeast and sheep erythrocytes are present in the plasma.