Substance P- and cholecytokinin-like immunoreactivity during post-metamorphic development of the central nervous system in the ascidian Ciona intestinalis

Following metamorphosis, the neural ganglion of ascidians is thought to be formed via the proliferation of epithelial cells comprising the ciliated duct. In adults, neuronal cell bodies expressing substance P- and gastrin/cholecystokinin-like immunoreactivity exhibit clearly defined patterns of distribution. Previous work shows that these patterns are re-established during regeneration of the adult ganglion. We have used antisera against substance P and cholecystokinin to monitor the formation of these patterns during normal post metamorphic development in Ciona intestinalis. Substance P cells first appear in the ganglion in animals of 1 mm body length. Cholecystokinin antiserum was not used at this stage but revealed a clear adult-like pattern of cells in the anterior region at the 3 to 5-mm stage. Substance P cells do not exhibit an adult pattern until animals have a body length of more than 10 mm. Proliferation in the neural complex was studied using the bromodeoxyuridine/anti-bromodeoxyuridine technique. Results suggest a mechanism whereby cells are born in the ciliated duct and later migrate to the ganglion. Double-labelling experiments indicate that more than 11 days elapse between cell birthdates and the expression of either of the peptides. Data presented suggest that the distributional patterns for these peptides during normal development are similar to those seen during regeneration.     

Distribution of GABA-like immunoreactivity during post-metamorphic development and regeneration of the central nervous system in the ascidian Ciona intestinalis

During regeneration of the neural ganglion in Ciona intestinalis, the pattern of reappearance of some peptidergic cells is similar to the ontogenetic patterns exhibited by these cell types during normal post-metamorphic development. Using a specific antiserum to gamma-aminobutyric acid (GABA), we describe here the appearance of GABA-ergic cells in Ciona during both post-metamorphic development and regeneration of the neural ganglion following total ablation. Post-metamorphic animals were divided into the categories: 1, 3–5, 6–10, 11–15 and 23–27 mm in body length. Regeneration was monitored at 12, 15, 18, 21, 28 and 56 days post ablation. The first appearance of GABA-like immunoreactive cells during normal development were at the 3 to 5-mm stage where they were seen as discrete cells, without processes, evenly distributed in the cortical region throughout the ganglion. Fibres were first seen at the 6 to 10-mm stage. As development proceeded, GABA-like immunoreactive cells became more concentrated near the nerve root exits and along the dorsal rind of the ganglion. In regenerating ganglia, GABA was first detected at 18–21 days post ablation, in cells that lacked any obvious processes and that were distributed in all regions of the ganglion. At 28 days post ablation, processes could be detected in the neuropile, and after 56 days GABA cells were found predominantly in the same regions as in the normally developing adult ganglion. Although the overall pattern reflects that in a normal adult, a few differences were detectable. For example, rather more GABAergic cells were concentrated ventrally in the ganglion close to the neural gland.     

Investigation of the contribution from peripheral GnRH-like immunoreactive 'neuroblasts' to the regenerating central nervous system in the protochordate Ciona intestinalis

The neural ganglion of the ascidian Ciona intestinalis regenerates in its entirety within a few weeks of ablation. Here we investigate the role of gonadotropin-releasing hormone-like immunoreactive (GnRH-li) cells in regeneration. Immunocytochemical studies show that in addition to a previously described plexus of GnRH-like neurones located in association with the dorsal strand, the normal adult brain contains GnRH-li neurones. These are predominantly localized to the ventral cortical rind at the posterior of the ganglion. Following ablation, non-process bearing GnRH-li cells appear in the regenerating area within two days. By day 8 post-ablation, process bearing GnRH-li cells are detected close to the regenerating brain. The number of these cells increases at later stages. GnRH-li cells are first detected within the regenerating brain at 14 days post-ablation and their number subsequently increases. These cells are initially concentrated along the ventral surface of the regenerating brain near to the dorsal strand. Double labelling studies with bromodeoxyuridine show that none of the GnRH-li cells are labelled at any stage of regeneration. The data are consistent with a sub-population of the new neurones being derived from GnRH-li neuroblasts born prior to ablation, which migrate from the dorsal strand complex into the regenerating ganglion.     

The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis

Phenoloxidase (PO) activity was examined in the tunic tissue of Ciona intestinalis following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the Dopa-MBTH reaction, displayed Ca(2+)-independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and THS was assayed for its PO activity. Analysis of variance of the time-course profiles showed that LPS was more effective in activating proPO. To disclose the PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. Microscopic observations and immunohistochemistry with anti-CinPO-2 antibodies showed granulocytes and unilocular refractile granulocytes containing PO, whereas few morula cells were stained. In THS zymograms (SDS-polyacrylamide gel electrophoresis), PO activity linked to 90-kDa and 120-kDa bands was observed as an effect of LPS injection, whereas the density of 170-kDa PO was weak. A third presumptive PO enzyme (CinPO-3) containing the CinPO-2 peptide was identified in the recent Ciona genome version. Presumably, LPS stimulated the production and dimerization (120 kDa) of CinPO-3 (66 kDa). Thus, the activated proPO system includes several POs that are distinguishable by size and that are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars.     

Effect of monosodium glutamate on the neural complex of Ciona intestinalis (Tunicata)

Summary Short-term treatment of the ascidian (tunicate) Ciona intestinalis with monosodium glutamate produces a transient decrease in methionine-enkephalin-like immunoreactivity of neurones in the nervous ganglion. Moreover, it causes vacuolisation of the cells in the neural complex, particularly in the neural gland. Similar damages occur after ovariectomy. These results suggest that the ovary exerts an indirect influence on the neural gland via the nervous ganglion, and that the methionine-enkephalin-like substance could be the responsible neuromediator.A portion of these results has been presented as a poster at the 10th International Symposium on Comparative Endocrinology, Copper Mountain, Colorado, USA (July 1985).     

Localization and enzymatic activity profiles of the proteases responsible for tachykinin-directed oocyte growth in the protochordate, Ciona intestinalis

We previously substantiated that Ci-TK, a tachykinin of the protochordate, Ciona intestinalis (Ci), triggered oocyte growth from the vitellogenic stage (stage II) to the post-vitellogenic stage (stage III) via up-regulation of the gene expression and enzymatic activity of the proteases: cathepsin D, carboxypeptidase B1, and chymotrypsin. In the present study, we have elucidated the localization, gene expression and activation profile of these proteases. In situ hybridization showed that the Ci-cathepsin D mRNA was present exclusively in test cells of the stage II oocytes, whereas the Ci-carboxypeptidase B1 and Ci-chymotrypsin mRNAs were detected in follicular cells of the stage II and stage III oocytes. Double-immunostaining demonstrated that the immunoreactivity of Ci-cathepsin D was largely colocalized with that of the receptor of Ci-TK, Ci-TK-R, in test cells of the stage II oocytes. Ci-cathepsin D gene expression was detected at 2h after treatment with Ci-TK, and elevated for up to 5h, and then slightly decreased. Gene expression of Ci-carboxypeptidase B1 and Ci-chymotrypsin was observed at 5h after treatment with Ci-TK, and then decreased. The enzymatic activities of Ci-cathepsin D, Ci-carboxypeptidase B1, and Ci-chymotrypsin showed similar alterations with 1-h lags. These gene expression and protease activity profiles verified that Ci-cathepsin D is initially activated, which is followed by the activation of Ci-carboxypeptidase B1 and Ci-chymotrypsin. Collectively, the present data suggest that Ci-TK directly induces Ci-cahtepsin D in test cells expressing Ci-TK receptor, leading to the secondary activation of Ci-chymotrypsin and Ci-carboxypeptidase B1 in the follicle in the tachykininergic oocyte growth pathway.     

Blue-green algalike cells associated with the tunic of Ciona intestinalis L.

Summary Certain organisms resembling blue-green algae embedded in the tunic of the solitary ascidian Ciona intestinalis L. are described. Their probable symbiotic role as related to the peculiar habitat of this ascidian is suggested.     

Regulatory roles of nitric oxide during larval development and metamorphosis in Ciona intestinalis

Metamorphosis in the ascidian Ciona intestinalis is a very complex process which converts a swimming tadpole to an adult. The process involves reorganisation of the body plan and a remarkable regression of the tail, which is controlled by caspase-dependent apoptosis. However, the endogenous signals triggering apoptosis and metamorphosis are little explored. Herein, we report evidence that nitric oxide (NO) regulates tail regression in a dose-dependent manner, acting on caspase-dependent apoptosis. An increase or decrease of NO levels resulted in a delay or acceleration of tail resorption, without affecting subsequent juvenile development. A similar hastening effect was induced by suppression of cGMP-dependent NO signalling. Inhibition of NO production resulted in an increase in caspase-3-like activity with respect to untreated larvae. Detection of endogenously activated caspase-3 and NO revealed the existence of a spatial correlation between the diminution of the NO signal and caspase-3 activation during the last phases of tail regression. Real-time PCR during development, from early larva to early juveniles, showed that during all stages examined, NO synthase (NOS) is always more expressed than arginase and it reaches the maximum value at late larva, the stage immediately preceding tail resorption. The spatial expression pattern of NOS is very dynamic, moving rapidly along the body in very few hours, from the anterior part of the trunk to central nervous system (CNS), tail and new forming juvenile digestive organs. NO detection revealed free diffusion from the production sites to other cellular districts. Overall, the results of this study provide a new important link between NO signalling and apoptosis during metamorphosis in C. intestinalis and hint at novel roles for the NO signalling system in other developmental and metamorphosis-related events preceding and following tail resorption.     

No chromosomal clustering of housekeeping genes in the marine chordate Ciona intestinalis

Housekeeping genes, widely expressed genes that are required for the basal function of most cell types, are clustered in the human and worm genomes. This arrangement suggests coordinate control of housekeeping gene expression at the chromosomal level. Here we examined whether this notion is applicable to a marine chordate, Ciona intestinalis. Using microarrays, we analyzed genes that were expressed in 11 organs of the adult, including the neural complex, branchial sac, esophagus, stomach, endostyle, intestine, body-wall muscle, heart, blood cells, ovary and testis. This analysis identified 158 genes that are expressed ubiquitously in these organs. These housekeeping genes could be classified into a range of Gene Ontology categories, in particular, ribosomal protein components. Of these 158 genes, we were able to map 141 genes onto the 14 pairs of the C. intestinalis chromosomes. They were distributed rather evenly over all the chromosomes, except for small clusters containing two or three genes. Therefore, the notion of chromosomal clustering of housekeeping genes is not applicable in this chordate.     

The occurrence of argyrophilic and argentaffin cells in the gut of Ciona intestinalis L.

Summary Argyrophilic and argentaffin cells occur in the stomach and intestinal epithelium of the sea-squirt, Ciona intestinalis L.. These cells are characterized by their basal swelling which contains the nucleus surrounded by small secretory granules and by a filamentous cell-apex which reaches the gut lumen. The cells are scattered unevenly within the epithelium. Their number decreases rapidly towards the lower part of the intestine. The localization, size of granules and their shape are features which differentiate these cells from other secretory cells in the gut epithelium such as mucous cells. These cells are thought to possess an endocrine function.The excellent technical assistance of Mrs. R. Sprang is gratefully acknowledged     

Pax gene expression in the developing central nervous system of Ciona intestinalis

We compare the expression patterns in Ciona intestinalis of three members of the Pax gene family, CiPax3/7, CiPax6 and Cipax2/5/8. All three genes are expressed in restricted patterns in the developing central nervous system. At the tailbud stage, CiPax3/7 is present in three patches in the brain and along the posterior neural tube, CiPax6 throughout the anterior brain and along the posterior neural tube and CiPax2/5/8 in a restricted region of the posterior brain. Double in situ hybridisations were used to identify areas of overlap between the expression of different genes. This showed that CiPax3/7 overlaps with the boundaries of CiPax6 expression in the anterior brain, and with CiPax2/5/8 in the posterior brain. The overlap between CiPax3/7 and CiPax2/5/8 is unlike that described in the ascidian Halocynthia rorezti.     

Substance P-, neurotensin- and bombesin-like immunoreactivities in the gill epithelium of Ciona intestinalis L.

Summary Substance P-, neurotensin- and bombesin-like immunoreactivities were localised in some gill epithelial cells in the pharynx of Ciona intestinalis L. No immunoreactivity was obtained with antisera to gastrin, glucagon, insulin, pancreatic polypeptide or calcitonin. Some of the epithelial cells of the gills were shown to be argyrophilic with the Grimelius technique.     

Localisation of somatostatin- and gastrin-like immunoreactivity in the gastrointestinal tract of Ciona intestinalis L.

Summary Somatostatin- and gastrin-like immunoreactivity has been found by immunofluorescence in cells of the stomach and intestinal epithelia of Ciona intestinalis L. The cells containing the peptide immunoreactive to mammalian anti-gastrin can be restained with the Grimelius' technique for argyrophilia.     

Structure and the evolutionary implication of the triplicated complement factor B genes of a urochordate ascidian, Ciona intestinalis

To elucidate the evolution of the complement system and MHC class III region, we analyzed the complement factor B (Bf) genes of a urochordate ascidian, Ciona intestinalis. Three different cDNA species, termed CiBf-1, CiBf-2 and CiBf-3, were identified. The deduced amino-acid sequences all contained the usual domains of vertebrate Bf and, in addition, three extra domains at the N-terminus. Furthermore, the serine protease domain of these CiBfs shared unique features with vertebrate complement components C1r/s and mannose-binding lectin-associated serine protease (MASP)-2/3, the absence of the disulfide bond designated histidine loop, and the usage of the AGY codon for the catalytic serine residue. These results indicate that complement genes have evolved through extensive exon shuffling events in the early stage of chordate evolution. Overall deduced amino-acid identity between CiBf-1 and -2 was 88%, whereas CiBf-3 showed 49% identity to both CiBf-1 and CiBf-2. These three CiBf genes were located within an approximately 50-kb genomic region, and exons 3 and 5 of all the three Bf genes showed an extremely high degree of nucleotide identity, indicating that the CiBf genes experienced extensive reorganization, such as duplication and gene conversion, since its divergence from the vertebrate Bf/C2 gene. Fluorescent in situ hybridization (FISH) to the chromosomes showed that genetic loci for the CiBfs, CiC3-1 and CiC3-2 genes are present on three different chromosomes, suggesting the possibility that the linkage among the MHC class III complement genes was established in the vertebrate lineage after its divergence from urochordates.The sequences reported in this paper have been deposited in the DDBJ database (accession nos. AB180044–AB180051).     

Inducible galectins are expressed in the inflamed pharynx of the ascidian Ciona intestinalis

Although ascidians belong to a key group in chordate phylogenesis, amino acid sequences of Ciona intestinalis galectin-CRDs (CiLgals-a and -b) have been retained too divergent from vertebrate galectins. In the present paper, to contribute in disclosing Bi-CRD galectin evolution a novel attempt was carried out on CiLgals-a and -b CRDs phylogenetic analysis, and their involvement in ascidian inflammatory responses was shown. CiLgals resulted aligned with Bi-CRD galectins from vertebrates (Xenopus tropicalis, Gallus gallus, Mus musculus, Homo sapiens), cephalochordates (Branchiostoma floridae), echinoderms (Strongylocentrotus purpuratus) and a mono-CRD galectin from the ascidian Clavelina picta. The CiLgals-a N-terminal and C-terminal CRDs contain the signature sequence involved in carbohydrate binding, whereas the CiLgals-b C-CRD presents only three out of seven key aminoacids and it could not be suitable as sugar binding motif. Sequence similarity between clusters suggests an evolutionary model based on CRD domain gene duplication and sequence diversification. In particular CiLgals-b N-CRD and C-CRD were similar to each other and both grouped with the ascidian C. picta mono-CRD. Homology modeling process shows a CiLgals molecular structure superimposed to chicken and mouse galectins. The CiLgals-a and CiLgals-b genes were upregulated by LPS inoculation suggesting that they are inducible and expressed in the inflamed pharynx as revealed by real-time PCR analysis. Finally, in situ hybridization and immunohistochemical assays showed their localization in the inflamed tissues, while immunoblotting analysis indicated that CiLgals can form oligomers.     

Localization of somatostatin-, substance P- and calcitonin-like immunoreactivity in the neural ganglion of Ciona intestinalis L. (Ascidiaceae)

Summary Indirect immunofluorescence studies using antisera to synthetic somatostatin, human calcitonin and substance P indicate, in the neural complex of the sea-squirt, Ciona intestinalis L., that these polypeptides are present in large perikarya situated at the periphery of the cerebral ganglion as well as in some smaller perikarya in the medulla. In the medullary and transitional zone, there are nerve fibres that cross-react positively with anti-calcitonin and antisubstance P.