Putative stem cells in the hemolymph and in the intestinal submucosa of the solitary ascidian Styela plicata.

BACKGROUND: In various ascidian species, circulating stem cells have been documented to be involved in asexual reproduction and whole-body regeneration. Studies of these cell population(s) are mainly restricted to colonial species. Here, we investigate the occurrence of circulating stem cells in the solitary Styela plicata, a member of the Styelidae, a family with at least two independent origins of coloniality. RESULTS: Using flow cytometry, we characterized a population of circulating putative stem cells (CPSCs) in S. plicata and determined two gates likely enriched with CPSCs based on morphology and aldehyde dehydrogenase (ALDH) activity. We found an ALDH + cell population with low granularity, suggesting a stem-like state. In an attempt to uncover putative CPSCs niches in S. plicata, we performed a histological survey for hemoblast-like cells, followed by immunohistochemistry with stem cell and proliferation markers. The intestinal submucosa (IS) showed high cellular proliferation levels and high frequency of undifferentiated cells and histological and ultrastructural analyses revealed the presence of hemoblast aggregations in the IS suggesting a possible niche. Finally, we document the first ontogenetic appearance of distinct metamorphic circulatory mesenchyme cells, which precedes the emergence of juvenile hemocytes. CONCLUSIONS: We find CPSCs in the hemolymph of the solitary ascidian Styela plicata, presumably involved in the regenerative capacity of this species. The presence of proliferating and undifferentiated mesenchymal cells suggests IS as a possible niche.

3-acetylpyridine-induced degeneration in the adult ascidian neural complex: Reactive and regenerative changes in glia and blood cells.

Ascidians are interesting neurobiological models because of their evolutionary position as a sister-group of vertebrates and the high regenerative capacity of their central nervous system (CNS). We investigated the degeneration and regeneration of the cerebral ganglion complex of the ascidian Styela plicata following injection of the niacinamide antagonist 3-acetylpyridine (3AP), described as targeting the CNS of several vertebrates. For the analysis and establishment of a new model in ascidians, the ganglion complex was dissected and prepared for transmission electron microscopy (TEM), routine light microscopy (LM), immunohistochemistry and Western blotting, 1 or 10 days after injection of 3AP. The siphon stimulation test (SST) was used to quantify the functional response. One day after the injection of 3AP, CNS degeneration and recruitment of a non-neural cell type to the site of injury was observed by both TEM and LM. Furthermore, weaker immunohistochemical reactions for astrocytic glial fibrillary acidic protein (GFAP) and neuronal ßIII-tubulin were observed. In contrast, the expression of caspase-3, a protein involved in the apoptotic pathway, and the glycoprotein CD34, a marker for hematopoietic stem cells, increased. Ten days after the injection of 3AP, the expression of markers tended toward the original condition. The SST revealed attenuation and subsequent recovery of the reflexes from 1 to 10 days after 3AP. Therefore, we have developed a new method to study ascidian neural degeneration and regeneration, and identified the decreased expression of GFAP and recruitment of blood stem cells to the damaged ganglion as reasons for the success of neuroregeneration in ascidians.

Regulation of nitric-oxide production in hemocytes of the ascidian Phallusia nigra.

Nitric oxide (NO) production in ascidians is related to immune responses of blood cells, and also to events such as egg fertilization and notochord regression. However, the signaling pathway for NO generation has been little investigated in this animal model. The present contribution identifies the cells involved in NO production and provides new information about a pathway for NO signaling. We were able to identify eight types of blood cells in the hemolymph of the ascidian Phallusia nigra, of which signet ring cells, univacuolar refractile granulocytes, and morula cells were involved in NO production. Zymosan A and lipopolysaccharide (LPS) enhanced NO production by blood cells, and the compound N-nitro-L-arginine-methyl ester (L-NAME) reduced NO production. Finally, the application of protein kinase A (PKA) and protein kinase C (PKC) inhibitors revealed that these molecules participate, together with NFκB, in the regulation of NO production by blood cells of P. nigra. This is the first report to show that PKA and PKC are involved in a signaling pathway that leads to NO production in ascidian blood cells.

Norepinephrine depresses the nitric oxide production in the ascidian hemocytes.

Norepinephrine (NE) is a neuro-hormone released by vertebrates and invertebrates during acute stress, and can influence their immune function. We found that NE depressed the production of nitric oxide (NO) by the hemocytes of ascidians. Our results with a fluorescent indicator for NO in assays using both NE and either α or ß-antagonist revealed that NE down-regulated NO production by the ascidian hemocytes. Our data suggest that NE may be acting via specific hemocyte receptors to induce a decrease in immune function.

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma.

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.

Collagen colocalizes with a protein containing a decorin-specific peptide in the tissues of the ascidian Styela plicata.

Decorin is an extracellular matrix dermatan sulfate/chondroitin sulfate proteoglycan found in a variety of vertebrate species. In the extracellular matrix of mammals, decorin interacts with fibrillar collagen and regulates its morphology. We report here the occurrence and distribution of collagen type I and the peptide, CEASGIGPEVPDDRD, which is present in the human decorin proteoglycan, in the extracellular matrix of different tissues of the primitive invertebrate chordate Styela plicata. The content of collagen was estimated by hydroxyproline, and its distribution in the tissues by histochemistry. Collagen was detected biochemically in intestine, heart, pharynx and mantle, occurring in higher amounts in the heart, followed by pharynx, mantle and intestine. Histochemical analysis with Sirius red indicates that collagen is present in the extracellular matrix of intestine and pharynx. Further ultrastructural immuno-gold assays using polyclonal antibodies raised against the decorin-specific peptide CEASGIGPEVPDDRD and collagen type I showed a co-localization of these molecules. These data suggest the occurrence of a protein containing a decorin-like peptide sequence, which may be interacting with fibrillar collagen in this primitive chordate.