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Dottorato di Ricerca in Biologia Animale, XXI Ciclo SSD,a.a.2007-2008 Università della Calabria

In Metazoa the maintenance of individuality and the defense against pathogens is devolved to the molecular mechanisms of the innate immunity. Adaptive immunity complements innate immunity in gnathostome vertebrates, thus indicating an increase of specialization of the defense mechanisms in the course of evolution. The Urochordate Ciona intestinalis, as the other invertebrates, does not have a gnathostome-type adaptive immune system, but, for its phylogenetic position, represents an excellent model for a comparative study of the elements and of the possible evolutionary pathways that led to the assembly of the adaptive system. As in other organisms, also in Ciona the blood cells have fundamental roles in immune defense, such as phagocytosis, encapsulation and release of substances with proinflammatory activity and in this context the understanding of their nature and function is fundamental. Despite the data collected in the last decades, many issues about blood cells in Ciona, as in other tunicates, remain still open. In particular, essential information about their embryonic and adult origin, their differentiation and even their precise classification, is still scanty, thus leaving the problem of blood cells genesis and homeostasis, also during immune reaction, unsolved. The project is aimed to the study of genes directly involved in hemocyte differentiation that may represent possible markers for the identification of hematopoietic sites. To this aim, genes that are involved in hematopoiesis in other organisms, such as Bmi, Ikaros, PU.1, Numb and GATA-a have been identified, sequenced, and characterized using PCR, in situ hybridization and immunochemistry during the post metamorphic stages and in the 4 adult tissues of C. intestinalis. Among these genes the C. intestinalis homolog of the Numb gene, involved in asymmetric cell division in mammals, has been identified as a possible marker for the study of hematopoiesis. The study of Ci-Numb provides preliminary information that helps in the understanding of hematopoietic processes of both the post-metamorphic and adult animal, and points out some previously undescribed aspects of the pharynx structure. In fact, the expression data produced at post-metamorphic stages and in the adult indicate that both the Ci-Numb transcripts and the protein are present in discrete regions of the animal body. These overlap with sites of proliferative activity and, more interestingly, with some of the regions previously indicated as hematopoietic. The morphological study at ultrastructural level of the tissues expressing Ci-Numb, in the juvenile as also in the adult, has allowed a more detailed characterization of the cell types that populate the hematopoietic sites. Morphological analysis of the structures of the adult pharynx, and of the stigmata in particular, show that the hematopoietic areas, described as compact groups of cells adjacent to the pharynx epithelium, correspond to the some cells that form the structure of the stigmas. This leads to a reassessment of the data that refer to the hematopoietic sites as “nodules” in the body wall adjacent to the muscle. The electron microscopy observations carried out in the juveniles demonstrate for the first time the genesis of the stigma. In conclusion, Ci-Numb can be considered a good marker of early hematopoiesis and may represent a baseline for further observations. Dottorat di Ricerca in Biologia Animale, XXIII Ciclo,a.a.2009-2010 Università della Calabria

Research over the past ten years indicates that the Complement System, the major effector arm of the vertebrate innate immunity, emerged at least 1,300 million years ago, long before the appearance of the adaptive immune response. These studies also pointed to the ascidian Ciona intestinalis (Urochordata), which occupies a key phylogenetic position in chordate evolution, as a suitable animal model for investigating the immune system evolution. In this context, two C3-like molecules, CiC3-1 and CiC3-2, exhibiting all the characteristic features of the mammalian C3s, have been previously identified, sequenced, and characterized in C. intestinalis. Furthermore, it has been found that the activation of CiC3-1 results in the release of the bioactive fragment C3-1a, a potent mediator of inflammatory reactions. The C3-1a fragment exerts its functional activity through the specific binding to a cell surface G protein-coupled seven-transmembrane receptor (CiC3aR), which has been more recently identified and characterized. Both C3-1a and its receptor are expressed only by granular and hyaline amoebocytes, two renewing blood cell types. In Ciona, blood cell proliferation occurs in circulating blood and in lymph nodules, mainly localized in the pharynx. In the present study I have first of all assessed whether Ciona C3 could represent an early molecular marker of amoebocyte proliferation in pharyngeal limph nodules. Using real-time PCR analysis I have found that CiC3-1 is constitutively expressed in the pharynx and that its expression is up-regulated by the treatment of the animals with lipopolysaccharides (LPS), a well-known inflammatory agent. By using two specific anti-C3 and anti-C3a polyclonal antibodies in immunohistological staining of pharynx sections, I have found that also the gene product is present in the pharynx, localized in areas identified as sites of hematogenic activity, and in amoebocytes occupying the pharyngeal bars. The same antibodies used in Western blot analysis indicated that CiC3-1 and its activation products, namely CiC3-1b and CiC3-1a, are present in homogenates of pharynx. The finding that the amount of the bioactive fragment CiC3-1a increases in the pharynx of LPS treated animals, clearly indicates that CiC3-1, present in the pharynx and labeling the maturing amoebocytes, is functionally active. The identification in C. intestinalis of both the partners participating in the complement-mediated inflammatory response gave me the opportunity for further investigating the C3a-C3aR interaction at both the molecular and cellular levels. In another series of experiments, I have focused my attention on cellular mechanisms acknowledged to be fundamental in ligand-receptor interaction. I have analyzed by confocal microscopy the dynamic process of internalization of the CiC3aR, induced by the binding of CiC3-1a in the form of the eighteen amino acid C-terminal peptide CiC3-1a59-76 of CiC3-1a fragment. These experiments have been performed on amoebocytes of circulating blood, which constitutively express both CiC3-1 and CiC3aR. Results of these experiments indicate that CiC3aR, following the agonist stimulus, is internalized in few minutes, and recycled to the cell surface within 30 min, made again available for CiC3-1a binding. My observations also revealed that in response to CiC3aR activation, Ciona arrestin (CiArr) is translocated to the plasma membrane in 30 sec. This localization profile is consistent with the participation of CiArr in CiC3aR internalization, and suggests, for the first time in Ciona, a role for CiArr as the agonist driven switch initiating receptor endocytosis. Dottorato di Ricerca in Biologia Animale, XXII Ciclo,a.a.2008-2009 Università della Calabria