A single oscillating proto-hypothalamic neuron gates taxis behavior in the primitive chordate Ciona.

Ciona larvae display a number of behaviors, including negative phototaxis. In negative phototaxis, the larvae first perform short spontaneous rhythmic casting swims. As larvae are cast in a light field, their photoreceptors are directionally shaded by an associated pigment cell, providing a phototactic cue. This then evokes an extended negative taxis swim. We report here that the larval forebrain of Ciona has a previously uncharacterized single slow-oscillating inhibitory neuron (neuron cor-assBVIN78) that projects to the midbrain, where it targets key interneurons of the phototaxis circuit known as the photoreceptor relay neurons. The anatomical location, gene expression, and oscillation of cor-assBVIN78 suggest homology to oscillating neurons of the vertebrate hypothalamus. Ablation of cor-assBVIN78 results in larvae showing extended phototaxis-like swims, even in the absence of phototactic cues. These results indicate that cor-assBVIN78 has a gating activity on phototaxis by projecting temporally oscillating inhibition to the photoreceptor relay neurons. However, in intact larvae, the frequency of cor-assBVIN78 oscillation does not match that of the rhythmic spontaneous swims, indicating that the troughs in oscillations do not themselves initiate swims but rather that cor-assBVIN78 may modulate the phototaxis circuit by filtering out low-level inputs while restricting them temporally to the troughs in inhibition.

Invertebrate Gonadotropin-Releasing Hormone Receptor Signaling and Its Relevant Biological Actions.

Gonadotropin-releasing hormones (GnRHs) play pivotal roles in reproduction via the hypothalamus-pituitary-gonad axis (HPG axis) in vertebrates. GnRHs and their receptors (GnRHRs) are also conserved in invertebrates lacking the HPG axis, indicating that invertebrate GnRHs do not serve as "gonadotropin-releasing factors" but, rather, function as neuropeptides that directly regulate target tissues. All vertebrate and urochordate GnRHs comprise 10 amino acids, whereas amphioxus, echinoderm, and protostome GnRH-like peptides are 11- or 12-residue peptides. Intracellular calcium mobilization is the major second messenger for GnRH signaling in cephalochordates, echinoderms, and protostomes, while urochordate GnRHRs also stimulate cAMP production pathways. Moreover, the ligand-specific modulation of signal transduction via heterodimerization between GnRHR paralogs indicates species-specific evolution in Ciona intestinalis. The characterization of authentic or putative invertebrate GnRHRs in various tissues and their in vitro and in vivo activities indicate that invertebrate GnRHs are responsible for the regulation of both reproductive and nonreproductive functions. In this review, we examine our current understanding of and perspectives on the primary sequences, tissue distribution of mRNA expression, signal transduction, and biological functions of invertebrate GnRHs and their receptors.

New approaches on the use of tunicates (Ciona robusta) for toxicity assessments.

After the accidental release of crude oil in marine environment, dispersants are applied on sea surface transferring oil into the water column where it can be broken down by biodegradation, thereby reducing potential pollution to coastal areas. Before they can be used in the wild, the ecotoxicity of such dispersants is usually evaluated with toxicity assays using algae, crustacean and fishes. Nowadays, there is a need to find alternative species to reduce the use of vertebrates both for ethical considerations and for reducing the cost of bioassays. Ciona robusta is a solitary ascidian that inhabits shallow waters and marine coastal areas. This species has been recently adopted as valuable biological model for ecotoxicity studies, thanks to its rapid embryonic and larval development, resemblance to vertebrates, and low risk of ethical issues. On this ground, the lethal and sublethal toxicity of two dispersants has been evaluated on Ciona juveniles. At this stage, the organisms become filter-feeders and the morphological alterations of the organs can be easily observed. The median lethal concentrations at 96 h (96hLC50) for Dispersant 1 (non-ionic surfactant) and for Dispersant 2 (mixture of non-ionic surfactants and anionic surfactants) are 41.6 mg/L (38.6-44.9) and 92.5 mg/L (87.7-97.5), respectively. The Ciona juvenile model was compared to Dicentrarchus labrax fish juveniles test, and it showed increased sensitivity for Ciona to these compounds. These results suggest that 96 h mortality test bioassay could be a good alternative method to 96 h mortality assay with D. labrax, limiting the use of vertebrates for dispersant toxicity.

Bioenergetic consequences from xenotopic expression of a tunicate AOX in mouse mitochondria: Switch from RET and ROS to FET.

Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc1 complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.

Teratogenic potential of nanoencapsulated vitamin A evaluated on an alternative model organism, the tunicate Ciona intestinalis.

Nano-encapsulation is a technology used to pack substances in order to enhance their stability and bioavailability, but this packing may interact with living systems, causing unexpected toxicity. Vitamin A (vit A) is a substance that has received attention, because in developed countries, the increasing availability of supplements is leading to its excessive intake. This study aims to compare teratogenic effects caused by exposure to the traditional formulation of vit A versus nano-encapsulated vit A. We used ascidian embryos as an alternative model. Ascidians are marine organisms closely related to vertebrates that share with them a body plan and developmental programme, including the morphogenetic role of retinoic acid (RA). Our data showed that the adverse effects of exposure to the same concentration of the two formulations were different, suggesting that the nano-encapsulation increased the bioavailability of the molecule, which could be better absorbed and metabolised to RA, the effective teratogenic substance.

Monoaminergic modulation of photoreception in ascidian: evidence for a proto-hypothalamo-retinal territory.

BACKGROUND: The retina of craniates/vertebrates has been proposed to derive from a photoreceptor prosencephalic territory in ancestral chordates, but the evolutionary origin of the different cell types making the retina is disputed. Except for photoreceptors, the existence of homologs of retinal cells remains uncertain outside vertebrates. METHODS: The expression of genes expressed in the sensory vesicle of the ascidian Ciona intestinalis including those encoding components of the monoaminergic neurotransmission systems, was analyzed by in situ hybridization or in vivo transfection of the corresponding regulatory elements driving fluorescent reporters. Modulation of photic responses by monoamines was studied by electrophysiology combined with pharmacological treatments. RESULTS: We show that many molecular characteristics of dopamine-synthesizing cells located in the vicinity of photoreceptors in the sensory vesicle of the ascidian Ciona intestinalis are similar to those of amacrine dopamine cells of the vertebrate retina. The ascidian dopamine cells share with vertebrate amacrine cells the expression of the key-transcription factor Ptf1a, as well as that of dopamine-synthesizing enzymes. Surprisingly, the ascidian dopamine cells accumulate serotonin via a functional serotonin transporter, as some amacrine cells also do. Moreover, dopamine cells located in the vicinity of the photoreceptors modulate the light-off induced swimming behavior of ascidian larvae by acting on alpha2-like receptors, instead of dopamine receptors, supporting a role in the modulation of the photic response. These cells are located in a territory of the ascidian sensory vesicle expressing genes found both in the retina and the hypothalamus of vertebrates (six3/6, Rx, meis, pax6, visual cycle proteins). CONCLUSION: We propose that the dopamine cells of the ascidian larva derive from an ancestral multifunctional cell population located in the periventricular, photoreceptive field of the anterior neural tube of chordates, which also gives rise to both anterior hypothalamus and the retina in craniates/vertebrates. It also shows that the existence of multiple cell types associated with photic responses predates the formation of the vertebrate retina.

Coupling of the phosphatase activity of Ci-VSP to its voltage sensor activity over the entire range of voltage sensitivity.

The voltage sensing phosphatase Ci-VSP is composed of a voltage sensor domain (VSD) and a cytoplasmic phosphatase domain. Upon membrane depolarization, movement of the VSD triggers the enzyme's phosphatase activity. To gain further insight into its operating mechanism, we studied the PI(4,5)P2 phosphatase activity of Ci-VSP expressed in Xenopus oocytes over the entire range of VSD motion by assessing the activity of coexpressed Kir2.1 channels or the fluorescence signal from a pleckstrin homology domain fused with green fluorescent protein (GFP) (PHPLC-GFP). Both assays showed greater phosphatase activity at 125 mV than at 75 mV, which corresponds to 'sensing' charges that were 90% and 75% of maximum, respectively. On the other hand, the activity at 160 mV (corresponding to 98% of the maximum 'sensing' charge) was indistinguishable from that at 125 mV. Modelling the kinetics of the PHPLC-GFP fluorescence revealed that its time course was dependent on both the level of Ci-VSP expression and the diffusion of PHPLC-GFP beneath the plasma membrane. Enzyme activity was calculated by fitting the time course of PHPLC-GFP fluorescence into the model. The voltage dependence of the enzyme activity was superimposable on the Q-V curve, which is consistent with the idea that the enzyme activity is tightly coupled to VSD movement over the entire range of membrane potentials that elicit VSD movement.

Expression of neuropeptide- and hormone-encoding genes in the Ciona intestinalis larval brain.

Despite containing only approximately 330 cells, the central nervous system (CNS) of Ciona intestinalis larvae has an architecture that is similar to the vertebrate CNS. Although only vertebrates have a distinct hypothalamus-the source of numerous neurohormone peptides that play pivotal roles in the development, function, and maintenance of various neuronal and endocrine systems, it is suggested that the Ciona brain contains a region that corresponds to the vertebrate hypothalamus. To identify genes expressed in the brain, we isolated brain vesicles using transgenic embryos carrying Ci-ß-tubulin(promoter)::Kaede, which resulted in robust Kaede expression in the larval CNS. The associated transcriptome was investigated using microarray analysis. We identified 565 genes that were preferentially expressed in the larval brain. Among these genes, 11 encoded neurohormone peptides including such hypothalamic peptides as gonadotropin-releasing hormone and oxytocin/vasopressin. Six of the identified peptide genes had not been previously described. We also found that genes encoding receptors for some of the peptides were expressed in the brain. Interestingly, whole-mount in situ hybridization showed that most of the peptide genes were expressed in the ventral brain. This catalog of the genes expressed in the larval brain should help elucidate the evolution, development, and functioning of the chordate brain.

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.

Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide.

To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.

Systematic analysis of embryonic expression profiles of zinc finger genes in Ciona intestinalis.

The recent decoding of a number of animal genomes has provided unprecedented information regarding evolution and gene structures, but this information must be supplemented with precise gene annotations and the temporal and spatial expression patterns of individual genes. In the present study, we systematically identified and characterized 566 zinc finger genes in the genome of Ciona intestinalis, an emerging model system for genome-wide studies of development and evolution. Of these genes, 356 genes encoded a potential transcription factor based on putative nucleic acid binding activity or domains of unknown function. We further examined the expression patterns of 225 genes during embryogenesis, and, when considered with a previous study [Imai, K.S., Hino, K., Yagi, K., Satoh, N., Satou, Y., 2004. Gene expression profiles of transcription factors and signaling molecules in the ascidian embryo: towards a comprehensive understanding of gene networks. Development 131, 4047-4058], we have characterized the developmental expression patterns of nearly 85% of the potential zinc finger-containing transcription factors. Overall, zinc finger genes are preferentially maternally expressed with little larval expression during development. The present study provides a valuable reference for genome-wide studies in this species and for future studies wishing to examine zinc finger gene expression patterns in other animals.

The dopamine-synthesizing cells in the swimming larva of the tunicate Ciona intestinalis are located only in the hypothalamus-related domain of the sensory vesicle.

Dopamine is a major neuromodulator synthesized by numerous cell populations in the vertebrate forebrain and midbrain. Owing to the simple organization of its larval nervous system, ascidian tunicates provide a useful model to investigate the anatomy, neurogenesis and differentiation of the dopaminergic neural network underlying the stereotypical swimming behaviour of its chordate-type larva. This study provides a high-resolution cellular analysis of tyrosine hydroxylase (TH)-positive and dopamine-positive cells in Ciona intestinalis embryos and larvae. Dopamine cells are present only in the sensory vesicle of the Ciona larval brain, which may be an ancestral chordate feature. The dopamine-positive cells of the ascidian sensory vesicle are located in the expression domain of homologues of vertebrate hypothalamic markers. We show here that the larval coronet cells also arise from this domain. As a similar association between coronet cells and the hypothalamus was reported in bony and cartilaginous fishes, we propose that part of the ascidian ventral sensory vesicle is the remnant of a proto-hypothalamus that may have been present in the chordate ancestor. As dopaminergic cells are specified in the hypothalamus in all vertebrates, we suggest that the mechanisms of dopamine cell specification are conserved in the hypothalamus of Ciona and vertebrates. To test this hypothesis, we have identified new candidate regulators of dopaminergic specification in Ciona based on their expression patterns, which can now be compared with those in vertebrates.

A saturation screen for cis-acting regulatory DNA in the Hox genes of Ciona intestinalis.

A screen for the systematic identification of cis-regulatory elements within large (>100 kb) genomic domains containing Hox genes was performed by using the basal chordate Ciona intestinalis. Randomly generated DNA fragments from bacterial artificial chromosomes containing two clusters of Hox genes were inserted into a vector upstream of a minimal promoter and lacZ reporter gene. A total of 222 resultant fusion genes were separately electroporated into fertilized eggs, and their regulatory activities were monitored in larvae. In sum, 21 separable cis-regulatory elements were found. These include eight Hox linked domains that drive expression in nested anterior-posterior domains of ectodermally derived tissues. In addition to vertebrate-like CNS regulation, the discovery of cis-regulatory domains that drive epidermal transcription suggests that C. intestinalis has arthropod-like Hox patterning in the epidermis.

FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells.

BACKGROUND: Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. RESULTS: One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. CONCLUSIONS: The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.

Regulatory gene expressions in the ascidian ventral sensory vesicle: evolutionary relationships with the vertebrate hypothalamus.

In extant chordates, the overall patterning along the anteroposterior and dorsoventral axes of the neural tube is remarkably conserved. It has thus been proposed that four domains corresponding to the vertebrate presumptive forebrain, midbrain-hindbrain transition, hindbrain, and spinal cord were already present in the common chordate ancestor. To obtain insights on the evolution of the patterning of the anterior neural tube, we performed a study aimed at characterizing the expression of regulatory genes in the sensory vesicle of Ciona intestinalis, the anteriormost part of the central nervous system (CNS) related to the vertebrate forebrain, at tailbud stages. Selected genes encoded primarily for homologues of transcription factors involved in vertebrate forebrain patterning. Seven of these genes were expressed in the ventral sensory vesicle. A prominent feature of these ascidian genes is their restricted and complementary domains of expression at tailbud stages. These patterning markers thus refine the map of the developing sensory vesicle. Furthermore, they allow us to propose that a large part of the ventral and lateral sensory vesicle consists in a patterning domain corresponding to the vertebrate presumptive hypothalamus.

Piecing together evolution of the vertebrate endocrine system.

One of the great challenges in biology is to understand how particular complex morphological and physiological characters originated in specific evolutionary lineages. In this article, we address the origin of the vertebrate hypothalamic-pituitary-peripheral gland (H-P-PG) endocrine system, a complex network of specialized tissues, ligands and receptors. Analysis of metazoan nucleotide and protein sequences reveals a patchwork pattern of H-P-PG gene conservation between vertebrates and closely related invertebrates (ascidians). This is consistent with a model of how the vertebrate H-P-PG endocrine system could have emerged in relatively few steps by gene family expansion and by regulatory and structural modifications to genes that are present in a chordate ancestor. Some of these changes might have resulted in new connections between metabolic or signaling pathways, such as the bridging of 'synthesis islands' to form an efficient system for steroid hormone synthesis.

A zinc finger transcription factor, ZicL, is a direct activator of Brachyury in the notochord specification of Ciona intestinalis.

In ascidian embryos, Brachyury is expressed exclusively in blastomeres of the notochord lineage and play an essential role in the notochord cell differentiation. The genetic cascade leading to the transcriptional activation of Brachyury in A-line notochord cells of Ciona embryos begins with maternally provided beta-catenin, which is essential for endodermal cell specification. beta-catenin directly activates zygotic expression of a forkhead transcription factor gene, FoxD, at the 16-cell stage, which in turn somehow activates a zinc finger transcription factor gene, ZicL, at the 32-cell stage, and then Brachyury at the 64-cell stage. One of the key questions to be answered is whether ZicL functions as a direct activator of Brachyury transcription, and this was addressed in the present study. A fusion protein was constructed in which a zinc finger domain of Ciona ZicL was connected to the C-terminus of GST. Extensive series of PCR-assisted binding site selection assays and electrophoretic mobility shift assays demonstrated that the most plausible recognition sequence of Ciona ZicL was CCCGCTGTG. We found the elements CACAGCTGG (complementary sequence: CCAGCTGTG) at -123 and CCAGCTGTG at -168 bp upstream of the putative transcription start site of Ci-Bra in a previously identified basal enhancer of this gene. In vitro binding assays indicated that the ZicL fusion protein binds to these elements efficiently. A fusion gene construct in which lacZ was fused with the upstream sequence of Ci-Bra showed the reporter gene expression exclusively in notochord cells when the construct was introduced into fertilized eggs. In contrast, fusion constructs with mutated ZicL-binding-elements failed to show the reporter expression. In addition, suppression of Ci-ZicL abolished the reporter gene expression, while ectopic and/or overexpression of Ci-ZicL resulted in ectopic reporter expression in non-notochord cells. These results provide evidence that ZicL directly activates Brachyury, leading to specification and subsequent differentiation of notochord cells.

The genome of the early chordate Ciona intestinalis encodes only five cytoplasmic intermediate filament proteins including a single type I and type II keratin and a unique IF-annexin fusion protein.

We screened the recently established draft genome of the early chordate Ciona intestinalis for genes encoding cytoplasmic intermediate filament (IF) proteins. The draft of the tunicate/urochordate genome contains only the five genes (IF-A, IF-B, IF-C, IF-D and IF-F) previously established by cDNA cloning. Three of these IF proteins (IF-D, IF-C, IF-A) were shown to be orthologs of vertebrate IF subfamilies I to III while two proteins (IF-B, IF-F) seemed tunicate specific. This is now firmly established for protein IF-F since the genomic data show that it arises as a fusion protein with a C-terminal annexin domain, a feature not found before in the very large collection of metazoan IF proteins. The results also confirm the previous proposal that urochordates lack orthologs of vertebrate type IV IF proteins. We discuss the striking increase of IF complexity from 5 tunicate to 65 human genes during chordate evolution. Thus the tunicate has a single keratin pair, which is expressed in the epidermis, while the human genome has at least 25 genes each for keratins I and keratins II. Finally there are four normal Ciona annexin genes in addition to the gene encoding the IF-annexin fusion proteins (IF-F).

Putative phenoloxidases in the tunicate Ciona intestinalis and the origin of the arthropod hemocyanin superfamily.

In addition to the respiratory copper-containing proteins for which it is named, the arthropod hemocyanin superfamily also includes phenoloxidases and various copperless storage proteins (pseudo-hemocyanins, hexamerins and hexamerin receptors). It had long been assumed that these proteins are restricted to the arthropod phylum. However, in their analysis of the predicted genes in the Ciona intestinalis (Urochordata:Tunicata) genome, Dehal et al. (Science 298:2157-2167) proposed that the sea squirt lacks hemoglobin but uses hemocyanin for oxygen transport. While there are, nevertheless, four hemoglobin genes present in Ciona, we have identified and cloned two cDNA sequences from Ciona that in fact belong to the arthropod hemocyanin superfamily. They encode for proteins of 794 and 775 amino acids, respectively. The amino acids required for oxygen binding and other structural important residues are conserved in these hemocyanin-like proteins. However, phylogenetic analyses and mRNA expression data suggest that the Ciona hemocyanin-like proteins rather act as phenoloxidases, possibly involved in humoral immune response. Nevertheless, the putative Ciona phenoloxidases demonstrate that the hemocyanin superfamily emerged before the Protostomia and Deuterostomia diverged and allow for the first time the unequivocal rooting of the arthropod hemocyanins and related proteins. Phylogenetic analyses using neighbor-joining and Bayesian methods show that the phenoloxidases form the most ancient branch of the arthropod proteins, supporting the idea that respiratory hemocyanins evolved from ancestors with an enzymatic function. The hemocyanins evolved in agreement with the expected phylogeny of the Arthropoda, with the Onychophora diverged first, followed by the Chelicerata and Pancrustacea. The position of the myriapod hemocyanins is not resolved.

Novel vanadium-binding proteins (vanabins) identified in cDNA libraries and the genome of the ascidian Ciona intestinalis.

Ascidians, especially those belonging to the suborder Phlebobranchia, can accumulate high levels of vanadium. Vanadium-binding proteins (vanabins) were first isolated from a vanadium-accumulating ascidian, Ascidia sydneiensis samea, and then the vanabins were cloned, their expression was studied, and metal-binding assays were conducted. In order to unravel the mechanism of vanadium accumulation, we searched for vanabin-like genes in other animals, including other ascidians. A database search revealed five groups of cDNAs that encoded vanabin-like proteins in another ascidian, Ciona intestinalis. The genes encoding C. intestinalis vanabins, CiVanabin1 to CiVanabin5, were clustered in an 8.4-kb genomic region. The direction of transcription of each gene was identical and each gene had a single intron. All the C. intestinalis vanabins were cysteine-rich, and the repetitive pattern of cysteines closely resembled that of A. sydneiensis samea vanabins. Using immobilized metal ion affinity chromatography (IMAC), we found that a recombinant protein of at least one of the C. intestinalis vanabins (CiVanabin5) bound to vanadium(IV) ions.