Rita Marino

Complement in urochordates: cloning and characterization of two C3-like genes in the ascidian Ciona intestinalis

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 α and β 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 α chain of CiC3-1 and CiC3-2, allowed the identification by Western blot of a single band in the blood serum, of about Mr 150,000. A phylogenetic tree, based on the alignment of CiC3-1 and CiC3-2 with molecules of the α2-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.

CiC3-1a-Mediated Chemotaxis in the Deuterostome Invertebrate Ciona intestinalis (Urochordata)

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 (CiC3-1a59–76) also promoted hemocyte chemotaxis. Furthermore, the CiC3-1a terminal Arg was not crucial for chemotactic activity, because the desArg peptide (CiC3-1a59–75) 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 Gi 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.

First Identification of a Chemotactic Receptor in an Invertebrate Species: Structural and Functional Characterization of Ciona intestinalis C3a Receptor

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.

A mutational approach to the study of development of the protochordate Ciona intestinalis (Tunicata, Chordata)

Abstract We have developed a protocol to perform a genetic screen for zygotic mutations affecting embryogenesis on the protochordate Ciona intestinalis. The choice of this taxon, whose phylogenetic position places it at the basis of the chordates as one the most primitive vertebrate relatives, could allow to address several evolutionary questions. The protochordates share many morphological features with the vertebrates, in primis the presence of a notochord. Ciona intestinalis shows several ideal features for a mutational analysis, such as external development and larvae made of a limited number of cells and cell types. Detailed cell lineage studies are available. The haploid genome size is comparable to the size of the Drosophila haploid genome. We have optimised conditions for chemical mutagenesis studying the efficiency at which different concentration of N-ethyl-N-nitrosourea (ENU) can induce mutations. Because the adult Ciona are hermaphrodites, we are performing a one-generation screen. The induced mutations are identified by visual inspection of developmental stages. We report the preliminary results from our screen including examples of the different classes of mutant phenotypes found so far.

Identification and developmental expression of Ci-isl, a homologue of vertebrate islet genes, in the ascidian Ciona intestinalis

Here we describe the cloning and expression pattern of Ci-isl, a homologue of vertebrate genes, in the ascidian. Early in development, Ci-isl expression occurs in the primordia of palps and brain vesicle, then in the tailbud embryo it is transiently extended to the notochord cells. At larva stage, the expression is down-regulated in the notochord, and it persists predominantly in the compartments of the nervous system. These observations indicate that also in invertebrates, islet genes show an expression pattern during differentiation of the nervous system.

Specific induction of self‐discrimination by follicle cells in Ciona intestinalis oocytes

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.

Evidence that metalloendoproteases are involved in gamete fusion of Ciona intestinalis, ascidia☆

The use of specific inhibitors and substrates of metalloendoproteases provides evidence that in many systems these enzymes are involved in membrane fusion events. In this study, we investigated whether metalloendoproteases are involved in Ciona sperm-egg fusion. In vitro fertilization assays with the metal chelator 1,10-phenanthroline, specific metalloendoprotease substrates, and the vital stain Hoechst 33342 suggested that a Zn(2+)-dependent metalloendoprotease(s) takes part in Ciona sperm-egg fusion. Furthermore, electrophysiological recordings showed that insemination carried out in the presence of either 1,10-phenanthroline or the substrate CBZ-Gly-Phe-NH2 fails to induce fertilization potential or any other change in membrane potential. These results support the hypothesis that in Ciona intestinalis, a metalloendoprotease(s) is functional in gamete fusion.

Ammonium channel expression is essential for brain development and function in the larva of Ciona intestinalis

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. J. Comp. Neurol. 503:135–147, 2007.

Further Observations on the Molecular Bases of Gamete Self-discrimination in Ciona intestinalis: Seasonal Variation of Self-sterility Rate

Ciona intestinalis oocytes are naturally self-sterile. However, self-sterility is not strict and occasionally self-fertile individuals can be observed during the main spawning season, that takes place in spring through early summer. Interestingly, the situation is completely reversed in the short period of autumn spawning. We know that the activity of Cihsp70, a member of the heat-inducible hsp gene family constitutively expressed exclusively in the accompanying follicle cells of Ciona intestinalis oocyte, is specifically involved in the acquisition of self-sterility. We have investigated whether a change in the expression of both Cihsp70 gene and protein could be responsible of this dramatic difference in the self-sterility rate.

Comparative localization of serotonin-like immunoreactive cells in Thaliacea informs tunicate phylogeny

BackgroundThaliaceans is one of the understudied classes of the phylum Tunicata. In particular, their phylogenetic relationships remain an issue of debate. The overall pattern of serotonin (5-HT) distribution is an excellent biochemical trait to interpret internal relationships at order level. In the experiments reported here we compared serotonin-like immunoreactivity at different life cycle stages of two salpid, one doliolid, and one pyrosomatid species. This multi-species comparison provides new neuroanatomical data for better resolving the phylogeny of the class Thaliacea.ResultsAdults of all four examined thaliacean species exhibited serotonin-like immunoreactivity in neuronal and non-neuronal cell types, whose anatomical position with respect to the nervous system is consistently identifiable due to α-tubulin immunoreactivity. The results indicate an extensive pattern that is consistent with the presence of serotonin in cell bodies of variable morphology and position, with some variation within and among orders. Serotonin-like immunoreactivity was not found in immature forms such as blastozooids (Salpida), tadpole larvae (Doliolida) and young zooids (Pyrosomatida).ConclusionsComparative anatomy of serotonin-like immunoreactivity in all three thaliacean clades has not been reported previously. These results are discussed with regard to studies of serotonin-like immunoreactivity in adult ascidians. Lack of serotonin-like immunoreactivity in the endostyle of Salpida and Doliolida compared to Pyrosomella verticillata might be the result of secondary loss of serotonin control over ciliary beating and mucus secretion. These data, when combined with other plesiomorphic characters, support the hypothesis that Pyrosomatida is basal to these clades within Phlebobranchiata and that Salpida and Doliolida constitute sister-groups.