Sea Squirt Ciona Intestinalis Research Articles

A microfluidic chip for immobilization and imaging of Ciona intestinalis larvae

AbstractSea squirts (Tunicata) are chordates and develop a swimming larva with a small and defined number of individually identifiable cells. This offers the prospect of connecting specific stimuli to behavioral output and characterizing the neural activity that links these together. Here, we describe the development of a microfluidic chip that allows live larvae of the sea squirt Ciona intestinalis to be immobilized and recorded. By generating transgenic larvae expressing GCaAMP6m in defined cells, we show that calcium ion levels can be recorded from immobilized larvae, while microfluidic control allows larvae to be exposed to specific waterborne stimuli. We trial this on sea water carrying increased levels of carbon dioxide, providing evidence that larvae can sense this gas.

Alternative oxidase gives Ciona colonists a head start in rotten egg water

Throughout the history of life on Earth, there have always been locations where some species thrive while others fail. And, as climate change and environmental degradation continue apace, some habitats are becoming more inhospitable as toxins such as hydrogen sulphide begin accumulating in zones when oxygen becomes scarce. The situation has rarely been so rosy for invasive creatures, such as the sea squirt Ciona intestinalis. But what might be giving these intruders the upper hand? Katharina Bremer from Tampere University, Finland, explains that the sea squirt is equipped with a specially adapted enzyme – known as ‘alternative oxidase’ – which allows them to produce the energy they require to survive in the presence of toxic hydrogen sulphide gas that smells of rotten eggs. The close relative of this critical enzyme – used by most creatures – is fatally poisoned by hydrogen sulphide. Could alternative oxidase prove to be the essential key that allows sea squirt invaders to colonise hydrogen sulphide hotspots where others perish?However, for pioneering sea squirts to gain a new foothold, they must send in a first wave of pilgrim settlers, and, in the case of the sea squirt, that means newly fertilized eggs and larvae. So, Bremer, Paul Debes (Hólar University, Iceland), Hitoyoshi Yasuo (Laboratoire de Biologie du Développement de Villefranche-sur-mer, France) and Howard Jacobs (Tampere University) needed to find out how toxic the dissolved gas is for freshly fertilized sea squirt eggs. After immersing the embryos in water ranging from 0 to 50 μmol l−1 hydrogen sulphide, which can prove fatal for fish, the team found that ∼80% of the youngsters successfully developed into larvae after 18.5 h in fresh seawater, with their survival rate falling to ∼60% when bathed in 10 μmol l−1 hydrogen sulphide. But then the youngsters began to struggle, with only ∼25% developing into larvae in 20 μmol l−1 hydrogen sulphide and none succeeding at 50 μmol l−1. Even with their specially adapted hydrogen-sulphide-proof alternative oxidise, the youngsters were only able to survive reasonably well in hydrogen sulphide concentrations of up to 15 μmol l−1.But then the team needed to check whether the embryos’ alternative oxidase provided them with their resistance to the toxin, or whether they were resorting to another strategy to survive the poison. The scientists injected the embryos with a molecule that prevented the youngsters from producing alternative oxidase, forcing the developing embryos to depend on other forms of the enzyme for survival in 15 μmol l−1 hydrogen sulphide. Without the key enzyme, their survival plummeted by 81% to 12%. However, when the team provided the impaired youngsters with an additional source of alternative oxidase, their survival rate returned to 27%; the same as normal sea squirt embryos in fresh seawater.In addition, the team measured whether the embryos were activating the gene responsible for the essential enzyme – and therefore likely to be producing the alternative oxidase – while developing in 15 μmol l−1 hydrogen sulphide, and it was clear that they had increased expression of the gene to combat the toxin. Their alternative oxidase source of energy is the key to the youngsters’ survival when their water reeks of rotten eggs. Bremer also warns that natural increases in hydrogen sulphide in waters across the globe that are becoming deoxygenated could provide other species that possess a hydrogen-sulphide-proof alternative oxidase with the advantage they require to go forth and colonise locations where other species can barely hang on.

Screening Gevis with Voltage Sensing Domains from Different Species of the Voltage Sensing Phosphatase Gene Family Reveals Differences in Membrane Expression and Voltage Ranges

Genetically encoded voltage indicators offer an opportunity to investigate the factors determining the voltage range of the voltage sensing phosphatase (VSP) gene family. The GEVI ArcLight utilizes a mutated form of the voltage sensing domain (VSD) from the sea squirt Ciona intestinalis to optically report membrane potentials in the physiological range since the wildtype VSD from Ciona exhibits a V1/2 (the membrane potential at which half of the total fluorescence change is observed) of +100 mV. A single mutation to the Ciona VSD changed the V1/2 to −30 mV for ArcLight. To investigate how nature determines the voltage range of a protein, we have generated several new GEVIs substituting the VSD from Ciona with other species ranging from mite to octopus to coelacanth to the zooplankton Eurytemora affinis. Most of these new GEVIs have similar V1/2 values to the Ciona wildtype VSD with some notable exceptions. The zooplankton GEVI had a V1/2 of +35 mV exhibiting a maximal fluorescence change of 18% ΔF/F for a 200 mV depolarization. No GEVI utilizing mammalian versions of the VSD from the phosphatase gene family yielded a signal due to poor plasma membrane expression suggesting the presence of a regulatory element in the mammalian VSP gene. Chimeras are now being generated in order to improve membrane expression and signal size.

Observations of a novel predatory gull behavior on an invasive ascidian: A new consequence of coastal urban sprawl?

Coastal urbanization has a dramatic effect on both terrestrial and marine ecosystems, altering resources such as food or space. Many species have shifted their ranges in response to anthropogenic pressures, resulting in novel species interactions. Here, we report an observation of a novel foraging behavior of the European Herring Gull (Larus argentatus): the capture and consumption of the widespread sea squirt Ciona intestinalis from under floating pontoons in a recreational marina in Ireland. Multiple gulls were observed performing a complex, multi‐step manipulation of several C. intestinalis individuals to remove their cellulose‐based tunic, which remained unconsumed. Further avenues of investigation are discussed, and hypotheses concerning possible ecosystem effects of novel ecological interactions occurring in proliferating artificial environments are presented.

Evolutionary origins of retinoid active short-chain dehydrogenases/reductases of SDR16C family

Vertebrate enzymes that belong to the 16C family of short-chain dehydrogenases/reductases (SDR16C) were shown to play an essential role in the control of retinoic acid (RA) levels during development. To trace the evolution of enzymatic function of SDR16C family, and to examine the origins of the pathway for RA biosynthesis from vitamin A, we identified putative SDR16C enzymes through the extensive search of available genome sequencing data in a subset of species representing major metazoan phyla. The phylogenetic analysis revealed that enzymes from protostome, non-chordate deuterostome and invertebrate chordate species are found in three clades of SDR16C family containing retinoid active enzymes, which are retinol dehydrogenase 10 (RDH10), retinol dehydrogenases E2 (RDHE2) and RDHE2-similar, and dehydrogenase reductase (SDR family) member 3 (DHRS3). For the initial functional analysis, we cloned RDH10- and RDHE2-related enzymes from the early developmental stages of a non-chordate deuterostome, green sea urchin Lytechinus variegatus, and an invertebrate chordate, sea squirt Ciona intestinalis. In situ hybridization revealed that these proteins are expressed in a pattern relevant to development, while assays performed on proteins expressed in mammalian cell culture showed that they possess retinol-oxidizing activity as their vertebrate homologs. The existence of invertebrate homologs of DHRS3 was inferred from the analysis of phylogeny and cofactor-binding residues characteristic of preference for NADP(H). The presence of invertebrate homologs in the DHRS3 group of SDR16C is interesting in light of the complex mutually activating interaction, which we have recently described for human RDH10 and DHRS3 enzymes. Further functional analysis of these homologs will establish whether this interaction evolved to control retinoid homeostasis only in vertebrates, or is also conserved in pre-vertebrates.

Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9.

The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, we report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. We use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the 'F0' generation revealed that endogenous Ebf function is required for specification of Islet-expressing motor ganglion neurons and atrial siphon muscles. We demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.

Ancient origin of mast cells

The sentinel roles of mammalian mast cells (MCs) in varied infections raised the question of their evolutionary origin. We discovered that the test cells in the sea squirt Ciona intestinalis morphologically and histochemically resembled cutaneous human MCs. Like the latter, C. intestinalis test cells stored histamine and varied heparin·serine protease complexes in their granules. Moreover, they exocytosed these preformed mediators when exposed to compound 48/80. In support of the histamine data, a C. intestinalis-derived cDNA was isolated that resembled that which encodes histidine decarboxylase in human MCs. Like heparin-expressing mammalian MCs, activated test cells produced prostaglandin D2 and contained cDNAs that encode a protein that resembles the synthase needed for its biosynthesis in human MCs. The accumulated morphological, histochemical, biochemical, and molecular biology data suggest that the test cells in C. intestinalis are the counterparts of mammalian MCs that reside in varied connective tissues. The accumulated data point to an ancient origin of MCs that predates the emergence of the chordates >500million years ago, well before the development of adaptive immunity. The remarkable conservation of MCs throughout evolution is consistent with their importance in innate immunity.

Symmetrical reproductive compatibility of two species in the Ciona intestinalis (Ascidiacea) species complex, a model for marine genomics and developmental biology.

The sea squirt Ciona intestinalis species complex is a widely used model system for genomics and developmental biology, as well as ecology. Contrary to previous reports, here we show no difference in the success of development and hatching between hybrid and conspecific crosses between the two species within this complex known as types A and B, from a region in the English Channel where they are sympatric. We grew laboratory hybrids in the field for three months, and successfully obtained reproductive adults. In back-crosses of F1 laboratory hybrids to parental types, normal larvae were obtained. We conclude that hybrid crosses generate viable offspring and the resulting hybrids are interfertile with types A and B. However we also show that introgression in the natural sympatric population remains low. We discuss possible pre-zygotic and post-zygotic mechanisms which reproductively isolate these species.

A Real Stretch: Mechanisms Behind Cell Elongation

A Real Stretch: Mechanisms Behind Cell Elongation

Identical sets of methylated and nonmethylated genes in Ciona intestinalis sperm and muscle cells

BackgroundThe discovery of gene body methylation, which refers to DNA methylation within gene coding region, suggests an as yet unknown role of DNA methylation at actively transcribed genes. In invertebrates, gene bodies are the primary targets of DNA methylation, and only a subset of expressed genes is modified.ResultsHere we investigate the tissue variability of both the global levels and distribution of 5-methylcytosine (5mC) in the sea squirt Ciona intestinalis. We find that global 5mC content of early developmental embryos is high, but is strikingly reduced in body wall tissues. We chose sperm and adult muscle cells, with high and reduced levels of global 5mC respectively, for genome-wide analysis of 5mC targets. By means of CXXC-affinity purification followed by deep sequencing (CAP-seq), and genome-wide bisulfite sequencing (BS-seq), we designated body-methylated and unmethylated genes in each tissue. Surprisingly, body-methylated and unmethylated gene groups are identical in the sperm and muscle cells. Our analysis of microarray expression data shows that gene body methylation is associated with broad expression throughout development. Moreover, transgenic analysis reveals contrasting gene body methylation at an identical gene-promoter combination when integrated at different genomic sites.ConclusionsWe conclude that gene body methylation is not a direct regulator of tissue specific gene expression in C. intestinalis. Our findings reveal constant targeting of gene body methylation irrespective of cell type, and they emphasize a correlation between gene body methylation and ubiquitously expressed genes. Our transgenic experiments suggest that the promoter does not determine the methylation status of the associated gene body.

Bacterial diversity associated with the tunic of the model chordate Ciona intestinalis

The sea squirt Ciona intestinalis is a well-studied model organism in developmental biology, yet little is known about its associated bacterial community. In this study, a combination of 454 pyrosequencing of 16S ribosomal RNA genes, catalyzed reporter deposition-fluorescence in situ hybridization and bacterial culture were used to characterize the bacteria living inside and on the exterior coating, or tunic, of C. intestinalis adults. The 454 sequencing data set demonstrated that the tunic bacterial community structure is different from that of the surrounding seawater. The observed tunic bacterial consortium contained a shared community of <10 abundant bacterial phylotypes across three individuals. Culture experiments yielded four bacterial strains that were also dominant groups in the 454 sequencing data set, including novel representatives of the classes Alphaproteobacteria and Flavobacteria. The relatively simple bacterial community and availability of dominant community members in culture make C. intestinalis a promising system in which to investigate functional interactions between host-associated microbiota and the development of host innate immunity.

The evolution and comparative neurobiology of endocannabinoid signalling.

CB(1)- and CB(2)-type cannabinoid receptors mediate effects of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide in mammals. In canonical endocannabinoid-mediated synaptic plasticity, 2-AG is generated postsynaptically by diacylglycerol lipase alpha and acts via presynaptic CB(1)-type cannabinoid receptors to inhibit neurotransmitter release. Electrophysiological studies on lampreys indicate that this retrograde signalling mechanism occurs throughout the vertebrates, whereas system-level studies point to conserved roles for endocannabinoid signalling in neural mechanisms of learning and control of locomotor activity and feeding. CB(1)/CB(2)-type receptors originated in a common ancestor of extant chordates, and in the sea squirt Ciona intestinalis a CB(1)/CB(2)-type receptor is targeted to axons, indicative of an ancient role for cannabinoid receptors as axonal regulators of neuronal signalling. Although CB(1)/CB(2)-type receptors are unique to chordates, enzymes involved in biosynthesis/inactivation of endocannabinoids occur throughout the animal kingdom. Accordingly, non-CB(1)/CB(2)-mediated mechanisms of endocannabinoid signalling have been postulated. For example, there is evidence that 2-AG mediates retrograde signalling at synapses in the nervous system of the leech Hirudo medicinalis by activating presynaptic transient receptor potential vanilloid-type ion channels. Thus, postsynaptic synthesis of 2-AG or anandamide may be a phylogenetically widespread phenomenon, and a variety of proteins may have evolved as presynaptic (or postsynaptic) receptors for endocannabinoids.

Genetic and genomic toolbox of the chordate Ciona intestinalis.

The experimental malleability and unique phylogenetic position of the sea squirt Ciona intestinalis as part of the sister group to the vertebrates have helped establish these marine chordates as model organisms for the study of developmental genetics and evolution. Here we summarize the tools, techniques, and resources available to the Ciona geneticist, citing examples of studies that employed such strategies in the elucidation of gene function in Ciona. Genetic screens, germline transgenesis, electroporation of plasmid DNA, and microinjection of morpholinos are all routinely employed, and in the near future we expect these to be complemented by targeted mutagenesis, homologous recombination, and RNAi. The genomic resources available will continue to support the design and interpretation of genetic experiments and allow for increasingly sophisticated approaches on a high-throughput, whole-genome scale.

The Yeast Complex I Equivalent NADH Dehydrogenase Rescues pink1 Mutants

Pink1 is a mitochondrial kinase involved in Parkinson's disease, and loss of Pink1 function affects mitochondrial morphology via a pathway involving Parkin and components of the mitochondrial remodeling machinery. Pink1 loss also affects the enzymatic activity of isolated Complex I of the electron transport chain (ETC); however, the primary defect in pink1 mutants is unclear. We tested the hypothesis that ETC deficiency is upstream of other pink1-associated phenotypes. We expressed Saccaromyces cerevisiae Ndi1p, an enzyme that bypasses ETC Complex I, or sea squirt Ciona intestinalis AOX, an enzyme that bypasses ETC Complex III and IV, in pink1 mutant Drosophila and find that expression of Ndi1p, but not of AOX, rescues pink1-associated defects. Likewise, loss of function of subunits that encode for Complex I–associated proteins displays many of the pink1-associated phenotypes, and these defects are rescued by Ndi1p expression. Conversely, expression of Ndi1p fails to rescue any of the parkin mutant phenotypes. Additionally, unlike pink1 mutants, fly parkin mutants do not show reduced enzymatic activity of Complex I, indicating that Ndi1p acts downstream or parallel to Pink1, but upstream or independent of Parkin. Furthermore, while increasing mitochondrial fission or decreasing mitochondrial fusion rescues mitochondrial morphological defects in pink1 mutants, these manipulations fail to significantly rescue the reduced enzymatic activity of Complex I, indicating that functional defects observed at the level of Complex I enzymatic activity in pink1 mutant mitochondria do not arise from morphological defects. Our data indicate a central role for Complex I dysfunction in pink1-associated defects, and our genetic analyses with heterologous ETC enzymes suggest that Ndi1p-dependent NADH dehydrogenase activity largely acts downstream of, or in parallel to, Pink1 but upstream of Parkin and mitochondrial remodeling.

MlRho – a program for estimating the population mutation and recombination rates from shotgun‐sequenced diploid genomes

Improvements in sequencing technology over the past 5 years are leading to routine application of shotgun sequencing in the fields of ecology and evolution. However, the theory to estimate evolutionary parameters from these data is still being worked out. Here we present an extension and implementation of part of this theory, mlRho. This program can efficiently compute the following three maximum likelihood estimators based on shotgun sequence data obtained from single diploid individuals: the population mutation rate (4N(e)mu), the sequencing error rate, and the population recombination rate (4N(e)c). We demonstrate the accuracy of mlRho by applying it to simulated data sets. In addition, we analyse the genomes of the sea squirt Ciona intestinalis and the water flea Daphnia pulex. Ciona intestinalis is an obligate outcrosser, while D. pulex is a cyclic parthenogen, and we discuss how these contrasting life histories are reflected in our parameter estimates. The program mlRho is freely available from http://guanine.evolbio.mpg.de/mlRho.

X-gal Staining of Electroporated Sea Squirt (Ciona) Embryos

INTRODUCTIONThis protocol describes the fixation, staining, and mounting of sea squirt (Ciona intestinalis) embryos and larvae that have been electroporated with a plasmid DNA containing a cis-regulatory DNA (e.g., tissue-specific enhancer) fused to the lacZ reporter gene. Green fluorescent protein (GFP) reporter genes can be directly visualized in living embryos and larvae, although fixed preparations can use aspects of this protocol.

A Genome May Reduce Your Carbon Footprint

A Genome May Reduce Your Carbon Footprint

An exceptional salt-tolerant antimicrobial peptide derived from a novel gene family of haemocytes of the marine invertebrate Ciona intestinalis

A novel gene family coding for putative antimicrobial peptides was identified in the EST (expressed sequence tag) database of the sea squirt Ciona intestinalis, and one of these genes was molecularly cloned from the Northern European Ciona subspecies. In situ hybridization and immunocytochemical analysis revealed that the natural peptide is synthesized and stored in a distinct haemocyte type, the univacuolar non-refractile granulocytes. By semiquantitative RT-PCR (reverse transcription-PCR) analysis, it was shown that the expression of the gene is markedly up-regulated in haemocytes after immune challenge. To evaluate the antimicrobial potency of the putative defence protein, we synthesized a peptide corresponding to its cationic core region. The peptide was highly effective against Gram-negative and Gram-positive bacteria including several human and marine pathogens as well as the yeast Candida albicans. Notably, the antibacterial activity of the peptide was retained at salt concentrations of up to 450 mM NaCl. Using two different methods we demonstrated that the peptide kills Gram-negative and Gram-positive bacteria by permeabilizing their cytoplasmic membranes. CD spectroscopy revealed that, in the presence of liposomes composed of negatively charged phospholipids, the peptide undergoes a conformational change and adopts an alpha-helical structure. Moreover, the peptide was virtually non-cytolytic for mammalian erythrocytes. Hence, the designed salt-tolerant antimicrobial peptide may represent a valuable template for the development of novel antibiotics.

Evolution of the bile salt nuclear receptor FXR in vertebrates*s⃞

Bile salts, the major end metabolites of cholesterol, vary significantly in structure across vertebrate species, suggesting that nuclear receptors binding these molecules may show adaptive evolutionary changes. We compared across species the bile salt specificity of the major transcriptional regulator of bile salt synthesis, the farnesoid X receptor (FXR). We found that FXRs have changed specificity for primary bile salts across species by altering the shape and size of the ligand binding pocket. In particular, the ligand binding pockets of sea lamprey (Petromyzon marinus) and zebrafish (Danio rerio) FXRs, as predicted by homology models, are flat and ideal for binding planar, evolutionarily early bile alcohols. In contrast, human FXR has a curved binding pocket best suited for the bent steroid ring configuration typical of evolutionarily more recent bile acids. We also found that the putative FXR from the sea squirt Ciona intestinalis, a chordate invertebrate, was completely insensitive to activation by bile salts but was activated by sulfated pregnane steroids, suggesting that the endogenous ligands of this receptor may be steroidal in nature. Our observations present an integrated picture of the coevolution of bile salt structure and of the binding pocket of their target nuclear receptor FXR.

The repertoire of G protein-coupled receptors in the sea squirt Ciona intestinalis

BackgroundG protein-coupled receptors (GPCRs) constitute a large family of integral transmembrane receptor proteins that play a central role in signal transduction in eukaryotes. The genome of the protochordate Ciona intestinalis has a compact size with an ancestral complement of many diversified gene families of vertebrates and is a good model system for studying protochordate to vertebrate diversification. An analysis of the Ciona repertoire of GPCRs from a comparative genomic perspective provides insight into the evolutionary origins of the GPCR signalling system in vertebrates.ResultsWe have identified 169 gene products in the Ciona genome that code for putative GPCRs. Phylogenetic analyses reveal that Ciona GPCRs have homologous representatives from the five major GRAFS (Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin) families concomitant with other vertebrate GPCR repertoires. Nearly 39% of Ciona GPCRs have unambiguous orthologs of vertebrate GPCR families, as defined for the human, mouse, puffer fish and chicken genomes. The Rhodopsin family accounts for ~68% of the Ciona GPCR repertoire wherein the LGR-like subfamily exhibits a lineage specific gene expansion of a group of receptors that possess a novel domain organisation hitherto unobserved in metazoan genomes.ConclusionComparison of GPCRs in Ciona to that in human reveals a high level of orthology of a protochordate repertoire with that of vertebrate GPCRs. Our studies suggest that the ascidians contain the basic ancestral complement of vertebrate GPCR genes. This is evident at the subfamily level comparisons since Ciona GPCR sequences are significantly analogous to vertebrate GPCR subfamilies even while exhibiting Ciona specific genes. Our analysis provides a framework to perform future experimental and comparative studies to understand the roles of the ancestral chordate versions of GPCRs that predated the divergence of the urochordates and the vertebrates.