Identificazione di C-Numb come marcatore dell'ematopoiesi di Ciona intestinalis

Dottorat di Ricerca in Biologia Animale, XXIII Ciclo,a.a.2009-2010In 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.Università della Calabri