ZEBRAFISH (DANIO RERIO) AS A MODEL FOR STUDYING CARDIOVASCULAR SYSTEM DEVELOPMENT. CHARACTERIZATION OF NEW MARKERS: VE-PTP, NUMB AND NUMBLIKE.

In the last years zebrafish (Danio rerio, Teleostea) has been well established as a great model organism for comparative studies of cardiovascular embryonic development in Teleostea and Vertebrate. A lot of processes are involved in the formation of a correct vascular system and we focalized our attention on the correct assembly of endothelial junctions and on the acquisition of the arterial-venous identity of endothelial cells. Association/disassociation of endothelial cell junctions plays a decisive role in controlling vascular development and blood vessels permeability. Ve-ptp (Vascular Endothelial-Protein Tyrosine Phosphatase) is a transmembrane protein exclusively present at endothelial adherens junctions (AJs) and its potential structural role in vascular integrity is not thoroughly described in vivo. In this work we investigated the role of Ve-ptp in AJs maturation/stability and in modulation of endothelial permeability. In fact, the performed ultrastructural analysis revealed a statistically significant reduction of junction complexes and the presence of immature AJs in zve-ptp morphants but not in control embryos. Moreover alteration of the zVe-ptp extracellular domain, induced by a splicing morpholino, resulted in permeability defects, showed by microangiography assays. Arterial-venous identity determination of endothelial cells represents a critical stage during cardiovascular development. Numb is a membrane-associated protein identified in all Vertebrates, while Numblike is a cytosolic protein, homologue to Numb, first characterized in mice. The functions of Numb and Numblike are related with their ability to physically interact with Notch, working as antagonist of this protein. Notch proteins are evolutionary conserved trans-membrane receptors, which regulate several mechanisms involved in developmental processes, including neurogenesis, haematopoiesis, vasculogenesis and angiogenesis. In this work we conducted a thorough functional characterization of numb and numblike in zebrafish embryos. As a consequence of loss-of-function experiments it was detected an evident reduction in size of caudal vascular plexus (CV) and an approach of the main axial vessels (Dorsal Aorta, DA, and Posterior Cardinal Vein, PCV), defects corroborated by morphological analysis of plastic semi-thin sections. Afterwards, expression pattern analysis of specific vascular markers revealed an increase of arterial markers’ expression and a decrease of venous markers’ expression, proving the alteration of AV identity acquisition of vessels. All these data taken together confirm a decisive role of ve-ptp, numb and numblike during vascular development in zebrafish.