IAP genes partake weighty roles in the astogeny and whole body regeneration in the colonial urochordate Botryllus schlosseri.

Inhibitors of Apoptosis Protein (IAP) genes participate in processes like apoptosis, proliferation, innate immunity, inflammation, cell motility, differentiation and in malignancies. Here we reveal 25 IAP genes in the tunicate Botryllus schlosseri's genome and their functions in two developmental biology phenomena, a new mode of whole body regeneration (WBR) induced by budectomy, and blastogenesis, the four-staged cycles of botryllid ascidian astogeny. IAP genes that were specifically upregulated during these developmental phenomena were identified, and protein expression patterns of one of these genes, IAP28, were followed. Most of the IAP genes upregulation recorded at blastogenetic stages C/D was in concert with the upregulation at 100 µM H2O2 apoptotic-induced treatment and in parallel to expressions of AIF1, Bax, Mcl1, caspase 2 and two orthologues of caspase 7. Wnt agonist altered the takeover duration along with reduced IAP expressions, and displacement of IAP28+ phagocytes. WBR was initiated solely at blastogenetic stage D, where zooidal absorption was attenuated and regeneration centers were formed either from remains of partially absorbed zooids or from deformed ampullae. Subsequently, bud-bearing zooids developed, in concert with a massive IAP28-dependent phagocytic wave that eliminated the old zooids, then proceeded with the establishment of morphologically normal-looking colonies. IAP4, IAP14 and IAP28 were also involved in WBR, in conjunction with the expression of the pro-survival PI3K-Akt pathway. IAPs function deregulation by Smac mimetics resulted in severe morphological damages, attenuation in bud growth and differentiation, and in destabilization of colonial coordination. Longtime knockdown of IAP functions prior to the budectomy, resulted in colonial death.

Acute exposure to water-soluble fractions of marine diesel oil: Evaluation of apoptosis and oxidative stress in an ascidian.

To understand the mechanisms involved in organisms' responses to toxicity from oil pollution, we studied the effect of acute exposure (24 h) to the marine water-soluble fraction of diesel oil (WFDO) on the ascidian Styela plicata. We evaluated the mortality and behavior by means of the siphon reflex, and the response of blood cells (hemocytes) contained in the pharynx, by means of the production of nitric oxide (NO) and reactive oxygen species (ROS), in addition to the activity of the antioxidant enzyme catalase (CAT). We also correlated oxidative stress with the activation of apoptotic pathways. No mortality occurred 24 h after the ascidians were exposed to 5% and 10% marine WFDO; however, the siphon reflex, a behavioral test based on the time that the animals took to close their siphons, increased. We also observed an inflammatory response, as estimated by the increase in the number of hemocytes in the pharynx. NO and ROS production and CAT activity were reduced, whereas caspase-3, a signaling molecule involved in apoptosis, was activated. This suggests that in ascidians acutely exposed to oil, another mechanism can occur in addition to oxidative stress. Another possibility is that WFDO may directly interact with cellular macromolecules and activate caspase-3, independently of generating oxidative stress. The results showed that components of diesel oil affected a marine organism, which showed reduced ROS production in the pharynx cells, including hemocytes, and activation of apoptotic pathways.

Ascidians: An Emerging Marine Model for Drug Discovery and Screening.

Ascidians (tunicates; sea squirts) are marine animals which provide a source of diverse, bioactive natural products, and a model for toxicity screenings. Compounds isolated from ascidians comprise an approved anti-tumor drug and many others are potent drug leads. Furthermore, the use of invertebrate embryos for toxicological screening tests or analysis offers the possibility to image a large number of samples for high throughput screens. Ascidians are members of a sister clade to the vertebrates and make a vertebrate-like tadpole larva composed of less than 3000 cells in 18 hours. The neural complex of the ascidian larva is made of only 350 cells (of which 100 are neurons) and functional genomic studies have now uncovered numerous GRNs underpinning neural specification and differentiation. Numerous studies showed that brain formation in ascidians is sensitive to toxic insults especially from endocrine disruptors making them a suitable model to study neurodevelopmental defects. Modern techniques available for ascidians, including transgenic embryos where 3D time lapse imaging of GFPexpressing reporter constructs can be analyzed, now permit numerous end-points to be evaluated in order to test the specific mode of action of many compounds. This review summarizes the key evidence suggesting that ascidian embryos are a favorable embryological model to study neurodevelopmental toxicity of different compounds with molecular and cellular end-points. We predict that ascidians may become a significant source of marine blue biotechnologies in the 21st century.

Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII.

The fertilising sperm triggers a transient Ca(2+) increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/C(cdc20) inhibitor, links release from metaphase II arrest with the Ca(2+) transient and its degradation is triggered by the Ca(2+)-induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca(2+) transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca(2+)-activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca(2+) increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates.

Influence of cadmium on the morphology and functionality of haemocytes in the compound ascidian Botryllus schlosseri.

In order to get insights into the effects of cadmium (Cd) on cell morphology and functions, we exposed haemocytes of the colonial ascidian Botryllus schlosseri to sub-lethal concentrations of CdCl(2). Results indicate that Cd hampers haemocyte spreading and phagocytosis in a dose-dependent way, through the alteration of the actin cytoskeleton. In addition, the metal decreases the stability of the internal membranes, as revealed by the Neutral Red assay. The fraction of cells showing positivity for the lysosomal enzyme acid phosphatase is also reduced in the presence of Cd, whereas the number of cells responsive to the Annexin-V assay and showing chromatin condensation increases, suggesting a metal-dependent induction of apoptosis in exposed cells. As Cd is a known cause of oxidative stress, the decrease in the percentage of cells positive to the assay for superoxide anion, observed at low Cd concentrations, is indicative of the synthesis of metal-chelating molecules, such as metallothioneins, whereas, the increase at high Cd concentrations suggests a depletion of the cell reducing redox potential.

Insight on signal transduction pathways involved in phagocytosis in the colonial ascidian Botryllus schlosseri.

Tunicates are chordate invertebrates, closely related to vertebrates, which represent valuable organisms for the study of a variety of biological processes from an evolutionary point of view. As invertebrates, they rely on innate immunity to cope with foreign, potentially pathogenic material. Among tunicates, the compound ascidian Botryllus schlosseri is emerging as a reliable model organism for the study of innate immune responses. However, there is a general lack of knowledge on the signalling pathways activated during immune responses and, in particular, in phagocytosis. In the present work, we carried out a preliminary investigation of the signalling pathways involved in phagocytosis, with particular reference to MAPK activation. We studied in vitro zymosan phagocytosis in the presence of manumycin A, which inhibit the activation of Ras, PD98059, SP600125 and SB202190, inhibitors of Erk, JNK and p38, respectively, parthenolide, N-acetyl-l-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), inhibiting NF-kB activation. In addition we carried out immunoblot and immunocytochemistry analysis with the use of anti-pErk1/2, anti-pp38, anti-pJNK, anti-NF-kB (p50) and anti-pan Ras antibodies. Results demonstrate that the recognition of foreign cells triggers a phosphorylation cascade leading to the activation of Ras-like small GTPases, MAPKs and NF-kB and argue in favour of a conservation, also in ascidians, of the main signalling pathways.

Role of Mos/MEK/ERK cascade and Cdk1 in Ca2+ oscillations in fertilized ascidian eggs.

Intracellular calcium ion concentration ([Ca(2+)](i)) transients are observed in the fertilized eggs of all species investigated so far, and are critical for initiating several events related to egg activation and cell cycle control. Here, we investigated the role of the Mos/MEK/ERK cascade and Cdk1 on Ca(2+) oscillations in fertilized ascidian eggs. The egg of the ascidian Phallusia nigra shows [Ca(2+)](i) oscillations after fertilization: Ca(2+) waves immediately following fertilization (phase I), and [Ca(2+)](i) oscillations between the first and second polar body extrusions (phase II). Our results show that in P. nigra eggs, ERK activity peaked just before the extrusion of the first polar body, and decreased gradually, eventually disappearing at the extrusion of the second polar body. Cyclin-dependent protein kinase 1(Cdk1) activity decreased to undetectable levels immediately after fertilization, and then periodically increased according to the meiotic and mitotic cell cycle. When the unfertilized eggs were incubated with U0126, an inhibitor of MEK, before insemination, ERK was immediately inactivated, and the phase II [Ca(2+)](i) oscillations disappeared. Alternatively, when the constitutively active Mos protein (GST-Mos) was injected into the unfertilized eggs, ERK activity was preserved for at least 120 min after fertilization, and the phase II [Ca(2+)](i) oscillations lasted for more than 120 min after the second polar body extrusion. These results suggest that ERK activity is necessary for maintaining [Ca(2+)](i) oscillations. GST-ΔN85-cyclin, which maintains Cdk1 activity, caused ERK activity in the eggs to persist for over 120 min after fertilization, and prolonged [Ca(2+)](i) oscillations. Moreover, the effects of GST-ΔN85-cyclin on the egg were abrogated by the application of U0126. Thus, Cdk1-mediated [Ca(2+)](i) oscillations seem to require ERK activity. However, GST-Mos triggered [Ca(2+)](i) oscillations after the second polar body extrusion, whereas GST-ΔN85-cyclin did not, although it prolongs the duration of [Ca(2+)](i) oscillations. Interestingly, GST-ΔN85-cyclin increased the frequency of [Ca(2+)](i) transients in the Mos-induced [Ca(2+)](i) oscillations after the extrusion of the second polar body. Thus, Cdk1 could maintain, but not activate, ERK and [Ca(2+)](i) oscillations. ERK activity and [Ca(2+)](i) oscillations seem to form a negative feedback loop which may be responsible for maintaining the meiotic period.

Conservation and divergence of chemical defense system in the tunicate Oikopleura dioica revealed by genome wide response to two xenobiotics.

BACKGROUND: Animals have developed extensive mechanisms of response to xenobiotic chemical attacks. Although recent genome surveys have suggested a broad conservation of the chemical defensome across metazoans, global gene expression responses to xenobiotics have not been well investigated in most invertebrates. Here, we performed genome survey for key defensome genes in Oikopleura dioica genome, and explored genome-wide gene expression using high density tiling arrays with over 2 million probes, in response to two model xenobiotic chemicals - the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) the pharmaceutical compound Clofibrate (Clo). RESULTS: Oikopleura genome surveys for key genes of the chemical defensome suggested a reduced repertoire. Not more than 23 cytochrome P450 (CYP) genes could be identified, and neither CYP1 family genes nor their transcriptional activator AhR was detected. These two genes were present in deuterostome ancestors. As in vertebrates, the genotoxic compound BaP induced xenobiotic biotransformation and oxidative stress responsive genes. Notable exceptions were genes of the aryl hydrocarbon receptor (AhR) signaling pathway. Clo also affected the expression of many biotransformation genes and markedly repressed genes involved in energy metabolism and muscle contraction pathways. CONCLUSIONS: Oikopleura has the smallest number of CYP genes among sequenced animal genomes and lacks the AhR signaling pathway. However it appears to have basic xenobiotic inducible biotransformation genes such as a conserved genotoxic stress response gene set. Our genome survey and expression study does not support a role of AhR signaling pathway in the chemical defense of metazoans prior to the emergence of vertebrates.

Molecular and expression analysis of the farnesoid X receptor in the urochordate Halocynthia roretzi.

The farnesoid X receptors (FXRs) are the major transcriptional regulators of bile salt synthesis in vertebrates. However, the structural conservation of invertebrate FXRs has only been studied for the major model organisms and studies on additional invertebrate FXRs are clearly required to obtain better resolution of FXR phylogeny and comparative developmental insights in chordates. In the present study, the cDNA encoding the farnesoid X receptor, HrFXR, was cloned from a marine invertebrate Halocynthia roretzi. The open reading frame of HrFXR encoded 688 amino acids including a longer N-terminal region and showed overall sequence identities of 28-41% to vertebrate and Ciona intestinalis FXRs. The N-terminal activation function 1 (AF-1) and hinge domains of HrFXR displayed relatively low identities (<20%), whereas the DNA-binding and ligand-binding domains showed relatively high (>73%) and intermediate (21-50%) identities, respectively. Based on a phylogenetic analysis, HrFXR belonged to a urochordate group, which was placed differently from vertebrate FXRα and FXRß subgroups. Real-time quantitative PCR analysis revealed that the HrFXR mRNA originated maternally and was highly expressed in adult gonads. Additionally, HrFXR mRNA levels in the gills and hepatopancreas showed significantly higher values in animals with soft tunic syndrome compared to those of normal individuals. Furthermore, direct injection of cholic acid significantly increased HrFXR transcript levels in vivo, although an expression vector containing HrFXR cDNA did not show a significant transactivation function in response to a well-known ligand for vertebrate FXR, GW4064, in HepG2 cells. These results suggest that the tunicate FXR has different structural and expressional characteristics compared to those of vertebrate FXRs.

Toxic effects of two pesticides, Imazalil and Triadimefon, on the early development of the ascidian Phallusia mammillata (Chordata, Ascidiacea).

Azole compounds are fungicides used in agriculture and in clinical area and are suspected to produce craniofacial malformations in vertebrates. Toxicity tests on sperm viability, fertilization and embryogenesis of the solitary ascidian Phallusia mammillata were performed to evaluate the effects of two azole derivatives, Imazalil and Triadimefon. Ascidian (Chordata, Ascidiacea) embryos and larvae could provide biological criteria for seawater quality standards because the larvae are lecitotrophic and have a short pelagic period, allowing to run the larval toxicity tests over a short period of time. Imazalil and Triadimefon proved to have strong consequences on P. mammillata. They could influence the reproductive rate of the animal exerting their effects at different levels: acting as spermiotoxic agents, inhibiting fertilization and impairing embryological development. Fertilization rate significantly decreased after 30min exposure of sperm to 25microM Imazalil (P<0.0001) and after exposure of both gametes to 50microM Imazalil (P<0.05) and 1mM Triadimefon (P<0.0001) as compared to controls. Malformations caused by exposure of embryos to both substances were dose dependent. Imazalil median teratogenic concentration (TC50 concentration, the concentration that resulted in 50% malformed larvae) value was 0.67microM and median lethal concentration (LC50, the concentration that resulted in 50% embryos dead before completing the development) value was 10.23microM while for Triadimefon TC50 value was 29.56 and LC50 value was 173.7microM. Larvae developed from embryos treated with Imazalil and Triadimefon showed alterations of the anterior structures of the trunk: papillary nerves and the anterior central nervous system failed to correctly differentiate, as showed by immunostaining with anti-beta-tubulin antibody. Comparing the anomalies caused by retinoic acid, reported in a previous study, it was possible to hypothesize that malformations induced by Imazalil and Triadimefon could be due to a perturbation of the endogenous retinoid content, as it has been proposed for mammals. Ascidians proved to be good models to study the toxic effects of pesticides since they offered both the convenience of working with an invertebrate species and the tissue sensitivity to chemical compound comparable to vertebrates.

Oxidative stress induces cytotoxicity during rejection reaction in the compound ascidian Botryllus schlosseri.

When genetically incompatible colonies of the compound ascidian Botryllus schlosseri contact each other, a rejection reaction occurs, characterised by the appearance of cytotoxic foci along the touching borders. In the course of this reaction, morula cells, a common haemocyte-type in ascidians, release their vacuolar content, mainly phenoloxidase and its polyphenol substrata, upon the recognition of soluble factors diffusing from the alien colony through the partially fused tunic. In a previous paper, we demonstrated the relationship between phenoloxidase and cytotoxicity. Here, we investigated the effects of superoxide dismutase, catalase and sorbitol (scavengers of superoxide anions, peroxides and hydroxyl radicals, respectively) on the cytotoxicity observed in haemocyte cultures incubated with heterologous blood plasma. Although the above compounds have no effects on morula cell degranulation and phenoloxidase activity, they suppress cell death, suggesting that oxidative stress plays a key role in in vitro cytotoxicity. In addition, sorbitol reduces the extent of the cytotoxicity occurring in the rejection reaction between incompatible colonies, which stresses the important role of hydroxyl radicals in this process. The observation of a decrease in total and non-protein thiols in haemocytes previously incubated with heterologous blood plasma fits the hypothesis of oxidative stress as the main cause of phenoloxidase-related cytotoxicity.

Botulinum ADP-ribosyltransferase C3 induces elevation of the vitelline coat of ascidian eggs.

Botulinum exoenzyme C3 ADP-ribosylates a 23 kDa protein of unfertilized eggs of the ascidian, Halocynthia roretzi. Microinjection of C3 into the eggs induced elevation of the egg vitelline coat. Co-injection of heparin or EGTA with C3 inhibited the inducing effect of C3. The vitelline coat of eggs which had been previously co-injected with heparin and C3 was elevated by addition of calcium ionophore, but not by insemination. C3 also induced an increased formation of inositol 1,4,5-triphosphate (IP3) in ascidian egg membranes. Thus the ADP-ribosylation of small GTP-binding protein by C3 seems to be responsible for elevation of the vitelline coat of ascidian eggs through IP3 formation and intracellular calcium mobilization.

The influence of tunichrome and other reducing compounds on tunic and fin formation in embryonic Ascidia callosa Stimpson.

Tunic in 46-hr-old Ascidia callosa larvae reared from dechorionated neurulae is either markedly reduced in thickness or absent altogether. The epidermis is fragile and cuticular fins fail to develop. Dechorionated neurulae treated with tunichrome and other reducing compounds (e.g., glutathione, ascorbate) show an enhancement in tunic formation and rudimentary fin development. UV absorbance spectra of extracts from unfertilized eggs, late tail-bud embryos, and tadpole larvae indicate that tunichrome may be present in all developmental stages. Experiments with neurulae in which the chorion was punctured with tungsten needles but not removed signify that the test cells are the most likely source of tunichrome. Results are consistent with the hypothesis that tunichrome is involved in the natural processes of tunic morphogenesis in ascidian embryos.

Segregation during ascidian embryogenesis of egg cytoplasmic information for tissue-specific enzyme development.

Cleavage-arrested embryos of the ascidian Ciona intestinalis were able to differentiate two tissue-specific enzymes-muscle acetylcholinesterase (EC 3.1.1.7) and brain pigment cell tyrosinase (EC 1.10.3.1). Cytochalasin B, colchicine, Colcemid, and podophyllotoxin were used as cleavage inhibitors at early embryonic stages up to the 64-cell stage. Only certain cells in the cleavage-arrested embryos developed these histochemically detectable enzymes, and this ability followed the cell lineage patterns for the two tissues. This result implies the presence of specific positional information in the egg cytoplasm that is differentially segregated during cleavage. There were distinct and separate puromycin and actinomycin D sensitivity periods for the occurrence of each enzyme during development of both normal and cleavage-arrested embryos. The segregated information is apparently neither the enzyme proteins nor RNA templates for enzyme synthesis, but is probably concerned with activation of appropriate genes.