Transcriptomic Analyses Reveal 2 and 4 Family Members of Cytochromes P450 (CYP) Involved in LPS Inflammatory Response in Pharynx of Ciona robusta.

Cytochromes P450 (CYP) are enzymes responsible for the biotransformation of most endogenous and exogenous agents. The expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, and regulation by cytokines and hormones. In recent years, Ciona robusta, one of the closest living relatives of vertebrates, has become a model in various fields of biology, in particular for studying inflammatory response. Using an in vivo LPS exposure strategy, next-generation sequencing (NGS) and qRT-PCR combined with bioinformatics and in silico analyses, compared whole pharynx transcripts from naïve and LPS-exposed C. robusta, and we provide the first view of cytochrome genes expression and miRNA regulation in the inflammatory response induced by LPS in a hematopoietic organ. In C. robusta, cytochromes belonging to 2B,2C, 2J, 2U, 4B and 4F subfamilies were deregulated and miRNA network interactions suggest that different conserved and species-specific miRNAs are involved in post-transcriptional regulation of cytochrome genes and that there could be an interplay between specific miRNAs regulating both inflammation and cytochrome molecules in the inflammatory response in C. robusta.

Expression and actions of corticotropin-releasing factor/diuretic hormone-like peptide (CDLP) and teneurin C-terminal associated peptide (TCAP) in the vase tunicate, Ciona intestinalis: Antagonism of the feeding response.

Teneurin C-terminal associated peptide (TCAP) is a neuropeptide that bears some structural similarity to the corticotropin-releasing factor (CRF) family of peptides. TCAP and CRF are both implicated in the regulation of stress-related behaviors, as established in rodent models. However, in vertebrates, both TCAP and CRF possess three additional paralogous forms making vertebrate models difficult to assess with respect to TCAP-CRF interaction. As a urochordate, this species possesses single homologs of TCAP and of a CRF/Diuretic-like peptide (CDLP) in the genome, thereby establishing Ciona intestinalis as an excellent model organism to examine the interaction of these peptide systems. However, the lack of C. intestinalis synthetic peptides and specific antisera has complicated experimentation. We, therefore, prepared synthetic versions of CDLP and TCAP to prepare specific antisera and to investigate their bioactivity in this species. To analyze stress-related behaviors, a novel behavioral assay was used to characterize different types of contraction-based behaviors, using buccal opening contractions, cloacal opening contractions, lateral contractions, longitudinal contractions and expulsions. Protein and mRNA expression data indicate that the mature versions of both peptides are present in a number of tissues. With respect to behavioral activity, both TCAP- and CDLP-treated animals had distinct contraction profiles under ambient conditions. Moreover, food stimulation tests revealed that whereas CDLP-treated animals displayed a strong expulsion behavior in response to feeding, TCAP-treated animals did not show this effect. These actions are consistent with previous studies done in vertebrates.

Spermiotoxicity of nickel nanoparticles in the marine invertebrate Ciona intestinalis (ascidians).

Nickel nanoparticles (Ni NPs) are increasingly used in modern industries as catalysts, sensors, and in electronic applications. Due to this large use, their inputs into marine environment have significantly increased; however, the potential ecotoxicological effects in marine environment have so far received little attention. In particular, little is known on the impact of NPs on gamete quality of marine organisms and on the consequences on fertility potential. The present study examines, for the first time, the impact of Ni NPs exposure on sperm quality of the marine invertebrate Ciona intestinalis (ascidian). Several parameters related with sperm status such as plasma membrane lipid peroxidation, mitochondrial membrane potential (MMP), intracellular pH, DNA integrity, and fertilizing ability were assessed as toxicity end points after exposure to different Ni NPs concentrations. Ni NPs generate oxidative stress that in turn induces lipid peroxidation and DNA fragmentation, and alters MMP and sperm morphology. Furthermore, sperm exposure to Ni NPs affects their fertilizing ability and causes developmental anomalies in the offspring. All together, these results reveal a spermiotoxicity of Ni NPs in ascidians suggesting that the application of these NPs should be carefully assessed as to their potential toxic effects on the health of marine organisms that, in turn, may influence the ecological system. This study shows that ascidian sperm represent a suitable and sensitive tool for the investigation of the toxicity of NPs entered into marine environment, for defining the mechanisms of toxic action and for the environmental monitoring purpose.

Evaluation of drug toxicity profiles based on the phenotypes of ascidian Ciona intestinalis.

In vivo toxicity evaluation using model organisms is an important step for the development of new drugs. Here, we report that Ciona intestinalis, a chordate invertebrate, is beneficial to drug toxicity evaluation for the following reasons: rapid embryonic and larval development, resemblance to vertebrates, ease of management, low cost, transparent body, and low risk of ethical issues. The dynamic phenotypic change of Ciona larvae during metamorphosis prompted us to examine the effect of cytotoxic drugs on its development by quantifying six toxicity endpoints: degenerated tail size, ampulla length, rotation of body axis, stomach size, heart rate, and body size. As a result, mitochondrial respiratory inhibitors, tubulin polymerization/depolymerization inhibitors, or DNA/RNA synthesis inhibitors showed distinct toxicity profiles against these six endpoints, but drugs with the same targets showed a similar toxicity profile in Ciona. Our results suggest Ciona is an effective animal model for profiling drug toxicity and exploring the mechanisms of drugs with unknown targets.

Reciprocal and dynamic polarization of planar cell polarity core components and myosin.

The Ciona notochord displays planar cell polarity (PCP), with anterior localization of Prickle (Pk) and Strabismus (Stbm). We report that a myosin is polarized anteriorly in these cells and strongly colocalizes with Stbm. Disruption of the actin/myosin machinery with cytochalasin or blebbistatin disrupts polarization of Pk and Stbm, but not of myosin complexes, suggesting a PCP-independent aspect of myosin localization. Wash out of cytochalasin restored Pk polarization, but not if done in the presence of blebbistatin, suggesting an active role for myosin in core PCP protein localization. On the other hand, in the pk mutant line, aimless, myosin polarization is disrupted in approximately one third of the cells, indicating a reciprocal action of core PCP signaling on myosin localization. Our results indicate a complex relationship between the actomyosin cytoskeleton and core PCP components in which myosin is not simply a downstream target of PCP signaling, but also required for PCP protein localization.

Characterization and transcription studies of a phytochelatin synthase gene from the solitary tunicate Ciona intestinalis exposed to cadmium.

The major thiol-containing molecules involved in controlling the level of intracellular ROS in eukaryotes, acting as a nonenzymatic detoxification system, are metallothioneins (MTs), glutathione (GSH) and phytochelatins (PCs). Both MTs and GSH are well-known in the animal kingdom. PC was considered a prerogative of the plant kingdom but, in 2001, a phytochelatin synthase (PCS) gene was described in the nematode Caenorhabditis elegans; additional genes encoding this enzyme were later described in the earthworm Eisenia fetida and in the parasitic nematode Schistosoma mansoni but scanty data are available, up to now, for Deuterostomes. Here, we describe the molecular characteristics and transcription pattern, in the presence of Cd, of a PCS gene from the invertebrate chordate Ciona intestinalis, a ubiquitous solitary tunicate and demonstrate the presence of PCs in tissue extracts. We also studied mRNA localization by in situ hybridization. In addition, we analyzed the behavior of hemocytes and tunic cells consequent to Cd exposure as well as the transcription pattern of the Ciona orthologous for proliferating cell nuclear antigen (PCNA), usually considered a proliferation marker, and observed that cell proliferation occurs after 96h of Cd treatment. This matches the hypothesis of Cd-induced cell proliferation, as already suggested by previous data on the expression of a metallothionein gene in the same animal.

Are reproduction impairments of free spawning marine invertebrates exposed to zero-valent nano-iron associated with dissolution of nanoparticles?

Studies were carried out to assess the effects of coating applied to zero-valent nano-iron (nZVI) on early life stage development of three key marine invertebrate species Mytilus galloprovincialis, Ciona intestinalis and Psammechinus milliaris. Embryo development was assessed following a 2-h exposure of the sperm to concentrations of two nZVIs of up to 10 mg l(-1) followed by in vitro fertilisation. Disruption of embryo development was most severe in sea squirts followed by mussel, while the urchin embryos were not significantly affected as compared with controls. An over twofold decrease in fertilisation success alongside significant delay in the embryo development was observed, and the effect was more severe with the coated form, possibly owing to its better colloidal stability. We provide in vitro evidence for the rapid dissolution (within 2 h) of nZVI in seawater to a degree that concentration of total solute Fe released from the coated ZVI particles exceeds safe limits of NOECs established for dissolved Fe.

Differential gene regulation by V(IV) and V (V) ions in the branchial sac, intestine, and blood cells of a vanadium-rich ascidian, Ciona intestinalis.

Ascidians are hyperaccumulators that have been studied in detail. Proteins and genes involved in the accumulation process have been identified, but regulation of gene expression related to vanadium accumulation remains unknown. To gain insights into the regulation of gene expression by vanadium in a genome-wide manner, we performed a comprehensive study on the effect of excess vanadium ions on a vanadium-rich ascidian, Ciona intestinalis, using a microarray. RT-PCR and enzyme activity assay were performed from the perspective of redox and accumulation of metal ions in each tissue. Glutathione metabolism-related proteins were significantly up-regulated by V(IV) treatment. Several genes involved in the transport of vanadium and protons, such as Nramp and V-ATPase, were significantly up-regulated by V(IV) treatment. We observed significant up-regulation of glutathione synthesis and degradation pathways in the intestine and branchial sac. In blood cells, expression of Ci-Vanabin4, glutathione reductase activity, glutathione levels, and vanadium concentration increased after V(IV) treatment. V(IV) treatment induced significant changes related to vanadium exclusion, seclusion, and redox pathways in the intestine and branchial sac. It also induced an enhancement of the vanadium reduction and accumulation cascade in blood cells. These differential responses in each tissue in the presence of excess vanadium ions suggest that vanadium accumulation and reduction may have regulatory functions. This is the first report on the gene regulation by the treatment of vanadium-rich ascidians with excess vanadium ions. It provided much information for the mechanism of regulation of gene expression related to vanadium accumulation.

Screening of ovarian steroidogenic pathway in Ciona intestinalis and its modulation after tributyltin exposure.

In this study, we have identified several ovarian steroids in Ciona with high similarity to vertebrate steroids and showed that cholesterol, corticosterone, dehydroepiandrosterone, estrone, estradiol-17beta, testosterone, pregnenolone, progesterone, have identical molecular spectra with vertebrate steroids. In addition, we have studied the effects of an endocrine disruptor (tributyltin: TBT) on these sex hormones and their precursors, ovarian morphology, and gene expression of some key enzymes in steroidogenic pathway in the ovary of Ciona. Ovarian specimens were cultured in vitro using different concentrations of TBT (10(-5), 10(-4) and 10(-3)M). Ethanol was used as solvent control. Gene expression analysis was performed for adrenodoxin (ADREN) and adrenodoxin reductase (ADOX) (mediators of acute steroidogenesis) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). These transcripts were detected and measured by quantitative (real-time) polymerase chain reaction (qPCR). Sex steroids and their precursors were identified and quantified by a gas chromatography-mass spectroscopy (GC-MS) method. Exposure of Ciona ovaries to TBT produced modulations (either increased or decreased) of sterols and sex steroid levels, whereas no significant differences in ADREN, ADOX or 17beta-HSD mRNA expression patterns were observed. Histological analysis shows that TBT produced several modifications on Ciona ovarian morphology that includes irregular outline of nuclear membrane, less compacted cytoplasm, in addition to test and granulosa cells that were detached from the oocyte membrane. Given that the ascidians represent very simple experimental models for the study of endocrine disruption by environmental contaminants, our findings provide excellent models for multiple identification and quantification of sex steroid and their precursors in biological samples exposed to endocrine-disrupting chemicals and for direct extrapolation of such effects across taxonomic groups and phyla. In addition, these results suggest that Cionaintestinalis may be a suitable species for molecular ecotoxicological studies and biomarker model for endocrine-disrupting effects in marine invertebrates.

Functional diversity of signaling pathways through G protein-coupled receptor heterodimerization with a species-specific orphan receptor subtype.

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in control of reproduction via a hypothalamic-pituitary-periphery endocrine system and nervous systems of not only vertebrates but also invertebrates. GnRHs trigger several signal transduction cascades via GnRH receptors (GnRHRs), members of the G protein-coupled receptor (GPCR) family. Recently, six GnRHs (tunicate GnRH [tGnRH]-3 to tGnRH-8) and four GnRHRs (Ciona intestinalis [Ci]-GnRHR1 to GnRHR-4), including a species-specific paralog, Ci-GnRHR4 (R4) regarded as an orphan receptor or nonfunctional receptor, were identified in the protochordate, C. intestinalis, which lacks the hypothalamic-pituitary system. Here, we show novel functional modulation of GnRH signaling pathways via GPCR heterodimerization. Immunohistochemical analysis showed colocalization of R1 and R4 in test cells of the ascidian ovary. The native R1-R4 heterodimerization was detected in the Ciona ovary by coimmunoprecipitation analysis. The heterodimerization in HEK293 cells cotransfected with R1 and R4 was also observed by coimmunoprecipitation and fluorescent energy transfer analyses. Binding assay revealed that R4 had no affinity for tGnRHs, and the heterodimerization did not alter the binding affinity of R1 to the ligands. The R1-R4 elicited 10-fold more potent Ca2+ mobilization than R1 exclusively by tGnRH-6, although R1-mediated cyclic AMP production was not affected by any of tGnRHs via the R1-R4 heterodimer. Moreover, the R1-R4 heterodimer potentiated translocation of both Ca2+-dependent protein kinase C-alpha (PKCalpha) by tGnRH-6 and Ca2+-independent PKCzeta by tGnRH-5 and tGnRH-6, eventually leading to the upregulation of extracellular signal-regulated kinase (ERK) phosphorylation compared with R1 alone. These results provide evidence that the species-specific GnRHR orphan paralog, R4, serves as an endogenous modulator for the fine-tuning of activation of PKC subtype-selective signal transduction via heterodimerization with R1 and that the species-specific GPCR heterodimerization, in concert with multiplication of tGnRHs and Ci-GnRHRs, participates in functional evolution of neuropeptidergic GnRH signaling pathways highly conserved throughout the animal kingdom.

Isolation and expression of a novel MBL-like collectin cDNA enhanced by LPS injection in the body wall of the ascidian Ciona intestinalis.

Collectins are a family of calcium-dependent lectins that are characterized by their collagen-like domains. Considerable interest has been focused on this class of proteins because of their ability to interact with components of the complement system activating a cascade of events responsible for the activation of the innate immune system. A differential screening between LPS-challenged and naïve Ciona intestinalis has been performed allowing the isolation of a full length cDNA encoding for a 221 AA protein. In silico analysis has shown that this polypeptide displays protein domains with similarities to mannose-binding lectins. A phylogenetic analysis suggested that C. intestinalis MBL has evolved early as a prototype of vertebrate MBL. Real-time PCR assay demonstrated that this gene is strongly activated after LPS injection in the tunica. In situ hybridization performed in LPS-induced animals has shown that this gene is expressed in granular amoebocytes and large granules hemocytes in the inflamed body wall tissue. Finally, an antimicrobial activity of the C. intestinalis MBL has been demonstrated.

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.

Molecular characterization of axonemal proteins and signaling molecules responsible for chemoattractant-induced sperm activation in Ciona intestinalis.

Spermatozoa undergo dramatic physiological changes at fertilization. In the ascidian Ciona intestinalis, an egg-derived substance named SAAF induces both sperm activation and chemotaxis to the egg. To elucidate the molecular mechanism underlying these phenomena, whole sperm proteins before and after SAAF-treatment were analyzed by two-dimensional gel electrophoresis. By comparison of spot patterns before and after activation, we found twelve proteins that changed the isoelectric points. Seven proteins were shown to be axonemal proteins and others were suggested to be non-axonemal components. Analysis of these proteins by MS-based proteomic system revealed that components of several substructures of the axonemes underwent the changes in isoelectric point at sperm activation, including WD-repeat intermediate chains of outer and inner arm dyneins and a radial spoke protein LRR37, as well as novel axonemal proteins with armadillo repeats or SMC domain. Molecules for cell signaling such as 14-3-3 proteins, Skp1 and VCP/p97 also showed isoelectric changes at sperm activation. These results show a comprehensive feature for signaling mechanism of the activation of spermatozoa at fertilization and also shed new lights on the regulation of ciliary and flagellar movements.

A conserved role for the MEK signalling pathway in neural tissue specification and posteriorisation in the invertebrate chordate, the ascidian Ciona intestinalis.

Ascidians are invertebrate chordates with a larval body plan similar to that of vertebrates. The ascidian larval CNS is divided along the anteroposterior axis into sensory vesicle, neck, visceral ganglion and tail nerve cord. The anterior part of the sensory vesicle comes from the a-line animal blastomeres, whereas the remaining CNS is largely derived from the A-line vegetal blastomeres. We have analysed the role of the Ras/MEK/ERK signalling pathway in the formation of the larval CNS in the ascidian, Ciona intestinalis. We show evidence that this pathway is required, during the cleavage stages, for the acquisition of: (1) neural fates in otherwise epidermal cells (in a-line cells); and (2) the posterior identity of tail nerve cord precursors that otherwise adopt a more anterior neural character (in A-line cells). Altogether, the MEK signalling pathway appears to play evolutionary conserved roles in these processes in ascidians and vertebrates, suggesting that this may represent an ancestral chordate strategy.

Induction of gamete release by gonadotropin-releasing hormone in a protochordate, Ciona intestinalis.

Gonadotropin-releasing hormone (GnRH) of vertebrates is now believed to have multiple functions in addition to its role as a hypophysiotropic hormone, as originally defined. Recently, it has been shown that GnRH occurs also in the ascidians, which are considered ancestral chordates. Here the author shows that GnRH induces spawning of gametes from mature individuals of Ciona intestinalis. Ciona accumulates mature gametes in the gonoducts and maintains them until spawning is triggered by a photoperiodic cue(s). Injection of synthetic tunicate GnRH-I or -II into various sites of mature individuals effectively induced gamete release (spawning), although the former was more potent. Gamete release often occurred on a larger scale than in spontaneous spawning. However, moderate gamete release, similar to spontaneous spawning, was often triggered by exogenous tunicate GnRH. GnRH in vivo apparently is released from the GnRH-containing neurons that are distributed from the region of the cerebral ganglion to the proximal part of the ovary along the dorsal strand within the blood sinus; this indicates that both forms of tunicate GnRH may be the actual inducers of spawning. It is suggested that, in the ancestral chordate, GnRH neurons release GnRH prior to the spawning and the released GnRH acts directly on the epithelium of gonoducts or functions as a neuromodulator of other neurons innervating the gonoducts to induce spawning.