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1.
Dev Genes Evol ; 230(4): 295-304, 2020 07.
Article in English | MEDLINE | ID: mdl-32632492

ABSTRACT

Earliest craniates possess a newly enlarged, elaborated forebrain with new cell types and neuronal networks. A key question in vertebrate evolution is when and how this cerebral expansion took place. The exon-junction complex (EJC) plays an essential role in mRNA processing of all Eukarya. Recently, it has been proposed that the EJC represses recursive RNA splicing in Deuterostomes, with implication in human brain diseases like microcephaly and depression. However, the EJC or EJC subunit contribution to brain development in non-vertebrate Deuterostomes remained unknown. Being interested in the evolution of chordate characters, we focused on the model species, Branchiostoma lanceolatum (Cephalochordata) and Ciona robusta (Tunicata), with the aim to investigate the ancestral and the derived expression state of Magoh orthologous genes. This study identifies that Magoh is part of a conserved syntenic group exclusively in vertebrates and suggests that Magoh has experienced duplication and loss events in mammals. During early development in amphioxus and ascidian, maternal contribution and zygotic expression of Magoh genes in various types of progenitor cells and tissues are consistent with the condition observed in other Bilateria. Later in development, we also show expression of Magoh in the brain of cephalochordate and ascidian larvae. Collectively, these results provide a basis to further define what functional role(s) Magoh exerted during nervous system development and evolution.


Subject(s)
Ciona intestinalis/genetics , Lancelets/genetics , Synteny/genetics , Animals , Ciona intestinalis/growth & development , Ciona intestinalis/metabolism , Lancelets/growth & development , Lancelets/metabolism , Nuclear Proteins/genetics
2.
Biol Open ; 7(1)2018 Jan 17.
Article in English | MEDLINE | ID: mdl-29222175

ABSTRACT

The gastrointestinal tract of Ciona intestinalis, a solitary tunicate that siphon-filters water, shares similarities with its mammalian counterpart. The Ciona gut exhibits other features that are unique to protochordates, including certain immune molecules, and other characteristics, e.g. chitin-rich mucus, which appears to be more widespread than considered previously. Exposure of Ciona to dextran sulphate sodium (DSS) induces a colitis-like phenotype similar to that seen in other systems, and is characterized by alteration of epithelial morphology and infiltration of blood cells into lamina propria-like regions. DSS treatment also influences the production and localization of a secreted immune molecule shown previously to co-localize to chitin-rich mucus in the gut. Resistance to DSS is enhanced by exposure to exogenous chitin microparticles, suggesting that endogenous chitin is critical to barrier integrity. Protochordates, such as Ciona, retain basic characteristics found in other more advanced chordates and can inform us of uniquely conserved signals shaping host-microbiota interactions in the absence of adaptive immunity. These simpler model systems may also reveal factors and processes that modulate recovery from colitis, the role gut microbiota play in the onset of the disease, and the rules that help govern the reestablishment and maintenance of gut homeostasis.

3.
Nat Commun ; 7: 10617, 2016 Feb 15.
Article in English | MEDLINE | ID: mdl-26875669

ABSTRACT

Protochordate variable region-containing chitin-binding proteins (VCBPs) consist of immunoglobulin-type V domains and a chitin-binding domain (CBD). VCBP V domains facilitate phagocytosis of bacteria by granulocytic amoebocytes; the function of the CBD is not understood. Here we show that the gut mucosa of Ciona intestinalis contains an extensive matrix of chitin fibrils to which VCBPs bind early in gut development, before feeding. Later in development, VCBPs and bacteria colocalize to chitin-rich mucus along the intestinal wall. VCBP-C influences biofilm formation in vitro and, collectively, the findings of this study suggest that VCBP-C may influence the overall settlement and colonization of bacteria in the Ciona gut. Basic relationships between soluble immunoglobulin-type molecules, endogenous chitin and bacteria arose early in chordate evolution and are integral to the overall function of the gut barrier.


Subject(s)
Carrier Proteins/immunology , Chitin/metabolism , Ciona intestinalis/immunology , Gastrointestinal Microbiome/immunology , Immunity, Mucosal/immunology , Immunoglobulin Variable Region/immunology , Intestinal Mucosa/immunology , Animals , Biofilms , Carrier Proteins/metabolism , Chitin Synthase/genetics , Chitin Synthase/metabolism , Immunohistochemistry , In Situ Hybridization, Fluorescence , Mucus
4.
Results Probl Cell Differ ; 57: 159-73, 2015.
Article in English | MEDLINE | ID: mdl-26537381

ABSTRACT

A variety of germline and somatic immune mechanisms have evolved in vertebrate and invertebrate species to detect a wide array of pathogenic invaders. The gut is a particularly significant site in terms of distinguishing pathogens from potentially beneficial microbes. Ciona intestinalis, a filter-feeding marine protochordate that is ancestral to the vertebrate form, possesses variable region-containing chitin-binding proteins (VCBPs), a family of innate immune receptors, which recognize bacteria through an immunoglobulin-type variable region. The manner in which VCBPs mediate immune recognition appears to be related to the development and bacterial colonization of the gut, and it is likely that these molecules are critical elements in achieving overall immune and physiological homeostasis.


Subject(s)
Bacteria/immunology , Ciona intestinalis/immunology , Gastrointestinal Tract/immunology , Receptors, Immunologic/immunology , Vertebrates/immunology , Animals , Biological Evolution , Ciona intestinalis/genetics , Ciona intestinalis/microbiology , Gastrointestinal Tract/metabolism , Gastrointestinal Tract/microbiology , Host-Pathogen Interactions/immunology , Immunity, Innate/genetics , Immunity, Innate/immunology , Receptors, Immunologic/genetics , Transcriptome/genetics , Transcriptome/immunology , Vertebrates/genetics , Vertebrates/microbiology
5.
PLoS One ; 9(5): e94984, 2014.
Article in English | MEDLINE | ID: mdl-24788831

ABSTRACT

Variable region-containing chitin-binding proteins (VCBPs) are secreted, immune-type molecules that have been described in both amphioxus, a cephalochordate, and sea squirt, Ciona intestinalis, a urochordate. In adult Ciona, VCBP-A, -B and -C are expressed in hemocytes and the cells of the gastrointestinal tract. VCBP-C binds bacteria in the stomach lumen and functions as an opsonin in vitro. In the present paper the expression of VCBPs has been characterized during development using in situ hybridization, immunohistochemical staining and quantitative polymerase chain reaction (qPCR) technologies. The expression of VCBP-A and -C is detected first in discrete areas of larva endoderm and becomes progressively localized during differentiation in the stomach and intestine, marking the development of gut tracts. In "small adults" (1-2 cm juveniles) expression of VCBP-C persists and VCBP-A gradually diminishes, ultimately replaced by expression of VCBP-B. The expression of VCBP-A and -C in stage 7-8 juveniles, at which point animals have already started feeding, is influenced significantly by challenge with either Gram-positive or -negative bacteria. A potential role for VCBPs in gut-microbiota interactions and homeostasis is indicated.


Subject(s)
Chitin/metabolism , Ciona intestinalis/physiology , Gastrointestinal Tract/microbiology , Gene Expression Regulation, Developmental , Host-Pathogen Interactions , Proteins/metabolism , Animals , Bacillus cereus/physiology , Ciona intestinalis/genetics , Ciona intestinalis/growth & development , Escherichia coli/physiology , Homeostasis , Microbiota , Protein Structure, Tertiary , Proteins/chemistry , Proteins/genetics , Stomach/microbiology
6.
PLoS One ; 9(4): e93386, 2014.
Article in English | MEDLINE | ID: mdl-24695540

ABSTRACT

It is now widely understood that all animals engage in complex interactions with bacteria (or microbes) throughout their various life stages. This ancient exchange can involve cooperation and has resulted in a wide range of evolved host-microbial interdependencies, including those observed in the gut. Ciona intestinalis, a filter-feeding basal chordate and classic developmental model that can be experimentally manipulated, is being employed to help define these relationships. Ciona larvae are first exposed internally to microbes upon the initiation of feeding in metamorphosed individuals; however, whether or not these microbes subsequently colonize the gut and whether or not Ciona forms relationships with specific bacteria in the gut remains unknown. In this report, we show that the Ciona gut not only is colonized by a complex community of bacteria, but also that samples from three geographically isolated populations reveal striking similarity in abundant operational taxonomic units (OTUs) consistent with the selection of a core community by the gut ecosystem.


Subject(s)
Ciona intestinalis/microbiology , Gastrointestinal Tract/microbiology , Microbiota/genetics , Animals , Bacteria/genetics , Ecosystem , Larva/microbiology , Metagenome/genetics , RNA, Ribosomal, 16S/genetics
7.
Front Immunol ; 3: 96, 2012.
Article in English | MEDLINE | ID: mdl-22563328

ABSTRACT

Complex symbiotic interactions at the surface of host epithelia govern most encounters between host and microbe. The epithelium of the gut is a physiologically ancient structure that is comprised of a single layer of cells and is thought to possess fully developed immunological capabilities. Ciona intestinalis (sea squirt), which is a descendant of the last common ancestor of all vertebrates, is a potentially valuable model for studying barrier defenses and gut microbial immune interactions. A variety of innate immunological phenomena have been well characterized in Ciona, of which many are active in the gut tissues. Interactions with gut microbiota likely involve surface epithelium, secreted immune molecules including variable region-containing chitin-binding proteins, and hemocytes from a densely populated laminar tissue space. The microbial composition of representative gut luminal contents has been characterized by molecular screening and a potentially relevant, reproducible, dysbiosis can be induced via starvation. The dialog between host and microbe in the gut can be investigated in Ciona against the background of a competent innate immune system and in the absence of the integral elements and processes that are characteristic of vertebrate adaptive immunity.

8.
Proc Natl Acad Sci U S A ; 108(40): 16747-52, 2011 Oct 04.
Article in English | MEDLINE | ID: mdl-21930927

ABSTRACT

A number of different classes of molecules function as structural matrices for effecting innate and adaptive immunity. The most extensively characterized mediators of adaptive immunity are the immunoglobulins and T-cell antigen receptors found in jawed vertebrates. In both classes of molecules, unique receptor specificity is effected through somatic variation in the variable (V) structural domain. V region-containing chitin-binding proteins (VCBPs) consist of two tandem Ig V domains as well as a chitin-binding domain. VCBPs are encoded at four loci (i.e., VCBPA-VCBPD) in Ciona, a urochordate, and are expressed by distinct epithelial cells of the stomach and intestine, as well as by granular amoebocytes present in the lamina propria of the gut and in circulating blood. VCBPs are secreted into the gut lumen, and direct binding to bacterial surfaces can be detected by immunogold analysis. Affinity-purified native and recombinant VCBP-C, as well as a construct consisting only of the tandem V domains, enhance bacterial phagocytosis by granular amoebocytes in vitro. Various aspects of VCBP expression and function suggest an early origin for the key elements that are central to the dialogue between the immune system of the host and gut microflora.


Subject(s)
Carrier Proteins/metabolism , Chitin/metabolism , Ciona intestinalis/immunology , Gastrointestinal Tract/metabolism , Gastrointestinal Tract/microbiology , Immunoglobulin Variable Region/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Carrier Proteins/genetics , Ciona intestinalis/genetics , Ciona intestinalis/microbiology , DNA Primers/genetics , Gene Components , Immunohistochemistry , Italy , Massachusetts , Molecular Sequence Data , Phagocytosis/immunology , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology
9.
Immunogenetics ; 61(6): 463-81, 2009 Jun.
Article in English | MEDLINE | ID: mdl-19404636

ABSTRACT

Two selected receptor genes of the immunoglobulin superfamily (IgSF), one CTX/JAM family member, and one poliovirus receptor-like nectin that have features of adhesion molecules can be expressed by Ciona hemocytes, the effectors of immunity. They can also be expressed in the nervous system (CTX/JAM) and in the ovary (nectin). The genes encoding these receptors are located among one set of genes, spread over Ciona chromosomes 4 and 10, and containing other IgSF members homologous to those encoded by genes present in a tetrad of human (1, 3 + X, 11, 21 + 19q) or bird chromosomes (1, 4, 24, 31) that include the leukocyte receptor complex. It is proposed that this tetrad is due to the two rounds of duplication that affected a single prevertebrate ancestral region containing a primordial leukocyte receptor complex involved in immunity and other developmental regulatory functions.


Subject(s)
Evolution, Molecular , Phylogeny , Receptors, Immunologic/genetics , Vertebrates/genetics , Amino Acid Sequence , Animals , Cell Adhesion Molecules/genetics , Chickens/genetics , Chromosome Mapping , Ciona intestinalis/embryology , Ciona intestinalis/genetics , Ciona intestinalis/growth & development , Female , Gene Expression Regulation, Developmental , Hemocytes/metabolism , Histocompatibility Antigens Class I/genetics , In Situ Hybridization , Junctional Adhesion Molecules , Leukocytes/immunology , Leukocytes/metabolism , Male , Molecular Sequence Data , Nectins , Receptors, Immunologic/classification , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Synteny , Urochordata/embryology , Urochordata/genetics , Urochordata/growth & development , Vertebrates/classification , Vertebrates/immunology
10.
Differentiation ; 76(3): 267-82, 2008 Mar.
Article in English | MEDLINE | ID: mdl-17924966

ABSTRACT

C-type lectins play an important role in the immune system and are part of a large superfamily that includes C-type lectin-like domain (CTLD)-containing proteins. Divergent evolution, acting on the CTLD fold, has generated the Ca2+-dependent carbohydrate-binding lectins and molecules, as the lectin-like natural killer (NK) receptors that bind proteins, rather than sugars, in a Ca(2+)-independent manner. We have studied ciCD94-1, a CTLD-containing protein from the tunicate Ciona intestinalis, which is a homolog of the CD94 vertebrate receptor that is expressed on NK cells and modulates their cytotoxic activity by interacting with MHC class I molecules. ciCD94-1 shares structural features with the CTLD-containing molecules that recognize proteins, suggesting that it could be located along the evolutionary pathway leading to the NK receptors. ciCD94-1 was up-regulated in response to inflammation induced by lipopolysaccharide (LPS) acting on a blood cell type present in both the tunic and circulating blood. Furthermore, an anti-ciCD94-1 antibody specifically inhibited the phagocytic activity of these cells. ciCD94-1 was also expressed during development in the larva and in the early stages of metamorphosis in structures related to the nervous system, and loss of its function affected the correct differentiation of these territories. These findings suggest that ciCD94-1 has different roles in immunity and in development, thus strengthening the concept of gene co-option during evolution and of an evolutionary relationship between the nervous and the immune systems.


Subject(s)
Ciona intestinalis/metabolism , Hemocytes/metabolism , Larva/metabolism , Lectins, C-Type/metabolism , Animals , Base Sequence , Blotting, Western , DNA Primers , In Situ Hybridization , Lectins, C-Type/genetics , Microscopy, Electron, Transmission , Phagocytosis , Reverse Transcriptase Polymerase Chain Reaction
11.
J Comp Neurol ; 503(1): 135-47, 2007 Jul 01.
Article in English | MEDLINE | ID: mdl-17480017

ABSTRACT

Ammonium uptake into the cell is known to be mediated by ammonium transport (Amt) proteins, which are present in all domains of life. The physiological role of Amt proteins remains elusive; indeed, loss-of-function experiments suggested that Amt proteins do not play an essential role in bacteria, yeast, and plants. Here we show that the reverse holds true in the tunicate Ciona intestinalis. The genome of C. intestinalis contains two AMT genes, Ci-AMT1a and Ci-AMT1b, which we show derive from an ascidian-specific gene duplication. We analyzed Ci-AMT expression during embryo development. Notably, Ci-AMT1a is expressed in the larval brain in a small number of cells defining a previously unseen V-shaped territory; these cells connect the brain cavity to the external environment. We show that the knockdown of Ci-AMT1a impairs the formation of the brain cavity and consequently the function of the otolith, the gravity-sensing organ contained in it. We speculate that the normal mechanical functioning (flotation and free movement) of the otolith may require a close regulation of ammonium salt(s) concentration in the brain cavity, because ammonium is known to affect both fluid density and viscosity; the cells forming the V territory may act as a conduit in achieving such a regulation.


Subject(s)
Brain/growth & development , Carrier Proteins/metabolism , Ciona intestinalis/metabolism , Ion Channels/metabolism , Quaternary Ammonium Compounds/metabolism , Animals , Biological Transport, Active/physiology , Brain/embryology , Brain/metabolism , Carrier Proteins/genetics , Ciona intestinalis/embryology , Ciona intestinalis/growth & development , Embryo, Nonmammalian , Gene Deletion , Gene Expression Regulation, Developmental , Ion Channels/genetics , Ion Transport/physiology , Larva/growth & development , Tissue Distribution
12.
J Immunol ; 177(6): 4132-40, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16951378

ABSTRACT

In mammals, the bioactive fragment C3a, released from C3 during complement activation, is a potent mediator of inflammatory reactions and exerts its functional activity through the specific binding to cell surface G protein-coupled seven-transmembrane receptors. Recently, we demonstrated a Ciona intestinalis C3a (CiC3a)-mediated chemotaxis of hemocytes in the deuterostome invertebrate Ciona intestinalis and suggested an important role for this molecule in inflammatory processes. In the present work, we have cloned and characterized the receptor molecule involved in the CiC3a-mediated chemotaxis and studied its expression profile. The sequence, encoding a 95,394 Da seven-transmembrane domain protein, shows the highest sequence homology with mammalian C3aRs. Northern blot analysis revealed that the CiC3aR is expressed abundantly in the heart and neural complex and to a lesser extent in the ovaries, hemocytes, and larvae. Three polyclonal Abs raised in rabbits against peptides corresponding to CiC3aR regions of the first and second extracellular loop and of the third intracellular loop react specifically in Western blotting with a single band of 98-102 kDa in hemocyte protein extracts. Immunostaining performed on circulating hemocytes with the three specific Abs revealed that CiC3aR is constitutively expressed only in hyaline and granular amoebocytes. In chemotaxis experiments, the Abs against the first and second extracellular loop inhibited directional migration of hemocytes toward the synthetic peptide reproducing the CiC3a C-terminal sequence, thus providing the compelling evidence that C. intestinalis expresses a functional C3aR homologous to the mammalian receptor. These findings further elucidate the evolutionary origin of the vertebrate complement-mediated proinflammatory process.


Subject(s)
Chemotaxis/immunology , Ciona intestinalis/immunology , Complement C3a/metabolism , Membrane Proteins/chemistry , Receptors, Complement/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Ciona intestinalis/metabolism , Gene Expression Profiling , Hemocytes/chemistry , Hemocytes/physiology , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Membrane Proteins/isolation & purification , Molecular Sequence Data , Receptors, Complement/biosynthesis , Receptors, Complement/genetics , Receptors, Complement/isolation & purification , Structure-Activity Relationship
13.
Mol Reprod Dev ; 73(4): 501-11, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16425233

ABSTRACT

Using the whole-cell voltage clamp technique, the electrical changes in oocyte and embryo plasma membrane were followed during different meiotic and developmental stages in Ciona intestinalis. We show, for the first time, an electrophysiological characterization of the plasma membrane in oocytes at the germinal vesicle (GV) stage with high L-type calcium (Ca2+) current activity that decreased through meiosis. Moreover, the absence of Ca2+ reduced germinal vesicle breakdown (GVBD), which is consistent with a role of Ca2+ currents in the prophase/metaphase transition. In mature oocytes at the metaphase I (MI) stage, Ca2+ currents decreased and then disappeared and sodium (Na+) currents first appeared remaining high up to the zygote stage. Intracellular Ca2+ release was higher in MI than in GV, indicating that Ca2+ currents in GV may contribute to fill the stores which are essential for oocyte contraction at fertilization. The fertilization current generated in Na+ free sea water was significantly lower than the control; furthermore, oocytes fertilized in the absence of Na+ showed high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, suggesting that signaling pathways that mediate first cleavage do not rely on ion current activities. At the 8-cell stage embryo, a resumption of Na+ current activity and conductance occurred, without a correlation with specific blastomeres. Taken together, these results imply: (i) an involvement of L-type Ca2+ currents in meiotic progression from the GV to MI stage; (ii) a role of Na+ currents during electrical events at fertilization and subsequent development; (iii) a major role of plasma membrane permeability and a minor function of specific currents during initial cell line segregation events.


Subject(s)
Calcium Channels/metabolism , Calcium/metabolism , Cell Differentiation/physiology , Ciona intestinalis/embryology , Ciona intestinalis/metabolism , Fertilization/physiology , Oocytes/metabolism , Sodium Channels/metabolism , Sodium/metabolism , Animals , Calcium Channels/physiology , Fertilization in Vitro , Ion Channel Gating/physiology , Membrane Potentials/physiology , Oocytes/cytology , Oogenesis/physiology , Sodium Channels/physiology , Time Factors
14.
Immunol Rev ; 198: 233-48, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15199966

ABSTRACT

Urochordates and cephalochordates do not have an adaptive immune system involving the somatic rearrangement of their antigen receptor genes. They do not have antigen-presenting molecules of the major histocompatibility complex (MHC)-linked class I and II types. In the absence of such a system, the status of their genes reflects perhaps a primitive pre-recombination-activating gene (RAG) stage that could suggest the pathway leading to the genesis of the T-cell receptor (TCR) and antibodies. In the genome of Ciona intestinalis, genes that encode molecules with membrane receptor features have been found among many members of the immunoglobulin superfamily (Igsf). They use the domains typical of vertebrate antigen receptors and class I and II: the V, and C1-like domains. These genes belong to two families with recognizable homologs in vertebrates: the junctional adhesion molecule (JAM)/cortical thymocyte marker of Xenopus (CTX) family and the nectin family. The human homologs of these genes segregate in a single unit of four paralogous segments on chromosomes 1q, 3q, 11p, and 21q. These regions contain nowadays several genes involved in the adaptive immune system, and some related members are present in the MHC paralogs as well. They also contain receptor-like genes without homologs in Ciona but with related members in the protostome Drosophila. It looks as if in Ciona one detects what looks like the 'fossil' of one group of genes bound to duplicate and give rise to many crucial elements of the adaptive immune system. The modern homologs of these JAM, CTX, and nectins are all or almost all virus receptors, and the hypothesis is formulated that this property was taken advantage of during evolution to participate in the elaboration of either or both the somatically generated antigen-recognizing receptors and the antigen-presenting molecules.


Subject(s)
Ciona intestinalis/immunology , Immunoglobulins/genetics , Animals , Chromosome Mapping , Ciona intestinalis/genetics , Homeodomain Proteins/genetics , Homeodomain Proteins/immunology , Immunity, Innate , Immunoglobulins/immunology , Killer Cells, Natural/immunology , Phylogeny , Receptors, Immunologic/genetics , Receptors, Immunologic/immunology
15.
J Immunol ; 171(10): 5521-8, 2003 Nov 15.
Article in English | MEDLINE | ID: mdl-14607959

ABSTRACT

Deuterostome invertebrates possess complement genes, and in limited instances complement-mediated functions have been reported in these organisms. However, the organization of the complement pathway(s), as well as the functions exerted by the cloned gene products, are largely unknown. To address the issue of the presence of an inflammatory pathway in ascidians, we expressed in Escherichia coli the fragment of Ciona intestinalis C3-1 corresponding to mammalian complement C3a (rCiC3-1a) and assessed its chemotactic activity on C. intestinalis hemocytes. We found that the migration of C. intestinalis hemocytes toward rCiC3-1a was dose dependent, peaking at 500 nM, and was specific for CiC3-1a, being inhibited by an anti-rCiC3-1a-specific Ab. As is true for mammalian C3a, the chemotactic activity of C. intestinalis C3-1a was localized to the C terminus, because a peptide representing the 18 C-terminal amino acids (CiC3-1a(59-76)) also promoted hemocyte chemotaxis. Furthermore, the CiC3-1a terminal Arg was not crucial for chemotactic activity, because the desArg peptide (CiC3-1a(59-75)) retained most of the directional hemocyte migration activity. The CiC3-1a-mediated chemotaxis was inhibited by pretreatment of cells with pertussis toxin, suggesting that the receptor molecule mediating the chemotactic effect is G(i) protein coupled. Immunohistochemical analysis with anti-rCiC3-1a-specific Ab and in situ hybridization experiments with a riboprobe corresponding to the 3'-terminal sequence of CiC3-1, performed on tunic sections of LPS-injected animals, showed that a majority of the infiltrating labeled hemocytes were granular amebocytes and compartment cells. Our findings indicate that CiC3-1a mediates chemotaxis of C. intestinalis hemocytes, thus suggesting an important role for this molecule in inflammatory processes.


Subject(s)
Chemotaxis, Leukocyte/immunology , Ciona intestinalis/immunology , Complement C3a/physiology , Amino Acid Sequence , Animals , Cell Migration Inhibition , Cell-Free System/immunology , Cell-Free System/metabolism , Chemotactic Factors/metabolism , Ciona intestinalis/genetics , Complement C3a/chemical synthesis , Complement C3a/genetics , Complement C3a/immunology , Escherichia coli/genetics , Escherichia coli/immunology , Escherichia coli/metabolism , Hemocytes/cytology , Hemocytes/immunology , Hemocytes/metabolism , Hemolymph/cytology , Hemolymph/immunology , Immune Sera/pharmacology , Inflammation/immunology , Inflammation/pathology , Lipopolysaccharides/pharmacology , Molecular Sequence Data , Pertussis Toxin/pharmacology , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemical synthesis , Recombinant Proteins/pharmacology
16.
Immunogenetics ; 55(8): 570-81, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14530883

ABSTRACT

Genome-wide sequence analysis in the invertebrate chordate, Ciona intestinalis, has provided a comprehensive picture of immune-related genes in an organism that occupies a key phylogenetic position in vertebrate evolution. The pivotal genes for adaptive immunity, such as the major histocompatibility complex (MHC) class I and II genes, T-cell receptors, or dimeric immunoglobulin molecules, have not been identified in the Ciona genome. Many genes involved in innate immunity have been identified, including complement components, Toll-like receptors, and the genes involved in intracellular signal transduction of immune responses, and show both expansion and unexpected diversity in comparison with the vertebrates. In addition, a number of genes were identified which predicted integral membrane proteins with extracellular C-type lectin or immunoglobulin domains and intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based activation motifs (ITAMs) (plus their associated signal transduction molecules), suggesting that activating and inhibitory receptors have an MHC-independent function and an early evolutionary origin. A crucial component of vertebrate adaptive immunity is somatic diversification, and the recombination activating genes (RAG) and activation-induced cytidine deaminase (AID) genes responsible for the Generation of diversity are not present in Ciona. However, there are key V regions, the essential feature of an immunoglobulin superfamily VC1-like core, and possible proto-MHC regions scattered throughout the genome waiting for Godot.


Subject(s)
Ciona intestinalis/genetics , Ciona intestinalis/immunology , Animals , Antigen Presentation/genetics , Cytokines/physiology , Genes, MHC Class I/physiology , Genes, MHC Class II/physiology , Genome , Immunity, Innate/genetics , Membrane Glycoproteins/physiology , Phagocytosis , Receptors, Cell Surface/physiology , Signal Transduction , Toll-Like Receptors
17.
Proc Natl Acad Sci U S A ; 100(1): 149-54, 2003 Jan 07.
Article in English | MEDLINE | ID: mdl-12518063

ABSTRACT

The sperm-activating and -attracting factor released from the eggs of the ascidians Ciona intestinalis and Ciona savignyi requires extracellular Ca(2+) for activating sperm motility and eliciting chemotactic behavior of the activated sperm toward the egg. Here, we show that modulators of the store-operated Ca(2+) channel, SK&F96365, Ni(2+), 2-aminoethoxydiphenylborane, and thapsigargin inhibit the chemotactic behavior of the ascidian sperm; on the other hand, blockers of voltage-dependent Ca(2+) channels did not inhibit the chemotaxis, even though they inhibited the sperm activation operated by voltage-dependent Ca(2+) channels. The blockers of store-operated Ca(2+) channels also inhibited the asymmetrical flagellar beating and turning movements of the ascidian sperm, which are typical signs of sperm chemotaxis. Depletion of internal Ca(2+) stores by thapsigargin induced capacitative Ca(2+) entry into the sperm, which was blocked by SK&F96365. These results suggest that the intracellular Ca(2+) concentration increase through the store-operated Ca(2+) channels induces asymmetrical flagellar movements to establish the chemotactic behavior of the sperm.


Subject(s)
Calcium Channel Blockers/pharmacology , Calcium Channels/physiology , Chemotaxis/physiology , Ciona intestinalis/physiology , Sperm Motility/physiology , Spermatozoa/physiology , Urochordata/physiology , Animals , Calcium Signaling/physiology , Male , Nickel/pharmacology , Nifedipine/pharmacology , Sperm Motility/drug effects , Spermatozoa/drug effects
18.
Immunogenetics ; 53(12): 1055-64, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11904683

ABSTRACT

The recent identification of complement components in deuterostome invertebrates has indicated the presence of a complement system operating via an alternative pathway in echinoderms and tunicates and via a MBL-mediated pathway thus far identified only in tunicates. Here, we report the isolation of two C3-like genes, CiC3-1 and CiC3-2, from blood cell total RNA of the ascidian Ciona intestinalis. The deduced amino acid sequences of both Ciona C3-like proteins exhibit a canonical processing site for alpha and beta chains, a thioester site with an associated catalytic histidine and a convertase cleavage site, thus showing an overall similarity to the other C3 molecules already characterized. Southern blotting analysis indicated that each gene is present as a single copy per haploid genome. In situ hybridization experiments showed that both CiC3-1 and CiC3-2 are expressed in one type of blood cell, the compartment cells. Two polyclonal antibodies, raised against two deduced peptide sequences in the alpha chain of CiC3-1 and CiC3-2, allowed the identification by Western blot of a single band in the blood serum, of about M(r)150,000. A phylogenetic tree, based on the alignment of CiC3-1 and CiC3-2 with molecules of the alpha(2)-macroglobulin superfamily, indicated that the Ciona C3s form a cluster with Halocynthia roretzi C3. The phylogenetic analysis also suggested that the duplication event from which the CiC3-1 and CiC3-2 genes originated occurred in the urochordate lineage after the separation of the Halocynthia and Ciona ancestor.


Subject(s)
Ciona intestinalis/genetics , Ciona intestinalis/immunology , Complement C3/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA, Complementary/genetics , In Situ Hybridization , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid , Species Specificity
19.
Dev Growth Differ ; 37(3): 287-291, 1995 Jun.
Article in English | MEDLINE | ID: mdl-37281519

ABSTRACT

Self-incompatibility, a mechanism that prevents self-fertilization in ascidians, is based on the ability of the oocyte vitelline coat to distinguish and accept only heterologous spermatozoa. In Ciona intestinalis self-discrimination is established during late oogenesis and is contributed or controlled by products of the overlying follicle cells. In this study we have further investigated the role of the follicle cells in the onset of self-discrimination by using in vitro maturation of ovarian oocytes deprived of the follicle cells and incubated with either autologous or heterologous follicle cells. Fertilization assays demonstrate that the action of the follicle cells is exerted even when they are detached from the vitelline coat and that only autologous follicle cells can promote the induction of self-sterility on the egg coat. Electron microscopy of the oocytes during maturation reveals that the switch from self-fertility to self-sterility is accompanied by the appearance of a thin electron-dense layer on the outer surface of the vitelline coat. We suggest that the formation of this layer is the result of the interaction between products of the follicle cells and the autologous vitelline coat.

20.
Dev Growth Differ ; 30(2): 147-158, 1988 Apr.
Article in English | MEDLINE | ID: mdl-37281575

ABSTRACT

In the ascidian Ciona intestinalis the species-specific interaction between the spermatozoon and the egg occurs between the vitelline coat (VC) of the egg and the plasma membrane of the apical part of the head of the spermatozoa. Concanavalin A (Con A)-binding sites are present on this area of the sperm surface. We used Con A to identify and isolate the spermatozoon plasma membrane components that may be involved in the interaction with the VC. These glycoproteins have been identified on SDS-PAGE of a sperm membrane fraction (SMF) enriched with the extermal proteins, after incubation of the gel with 3 H-Con A. Affinity chromatography on Con A-agarose has been used for the purification of sperm plasma membrane proteins with and affinity for the lectin. The biological activity of the Con A-retained fraction was determined with binding and fertilization assays.

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