ZEBRAFISH AS A MODEL TO STUDY THE ROLE OF PAX5/ETV6 FUSION GENE IN ACUTE LYMPHOBLASTIC LEUKEMIA AND THE FUNCTION OF THE SINGLE WILD TYPE GENES IN NORMAL HEMATOPOIESIS

B cell precursors Acute Lymphoblastic Leukemia (BCP-ALL) is a tumor characterized by the malignant expansion of B cell progenitors and it is the most common childhood cancer. About 30% of BCP-ALL cases are characterized by mutations, deletions or translocations of the PAX5 gene. Among PAX5 translocations, the most recurrent is t(9;12) which determines the generation of PAX5/ETV6 fusion gene. The PAX5 transcription factor is essential for B cell lineage commitment and differentiation. PAX5 fulfills its function mainly through the activation of B lineage-specific genes and the repression of inappropriate ones. ETV6 transcription factor is specifically required for adult hematopoiesis in mice. Moreover, ETV6 accelerates erythroid differentiation of cell lines and it stimulates hemoglobin synthesis in adult mice. PAX5/ETV6 is able to down-regulate the expression of some PAX5-activated-genes, as well as to up-regulate some PAX5-repressed-genes. Accordingly, PAX5/ETV6 caused the alteration of B cell differentiation and conferred survival advantages to mouse B cell precursors in vitro. The acquisition of survival advantages by PAX5/ETV6 expressing cells is due to the activation of the Lck-STAT5 pathway leading to the upregulation of STAT5 effectors Ccnd2 and cMyc. When we started this project very little was known about the conservation of pax5 and etv6 roles in zebrafish hematopoiesis. We thus analyzed their expression pattern and we found that these genes are expressed in several hematopoietic tissues. Indeed, pax5 is expressed in pancreas, which has been proposed to be a transient embryonic site of B cell lymphopoiesis in zebrafish. pax5 knockdown caused the alteration of the expression of several B cell markers. Otherwise, etv6 is expressed in tissues related to both primitive and definitive hematopoiesis. etv6 loss of function determined the reduction of primitive mature erythrocytes and we found that this alteration is due to the defective differentiation of primitive erythrocytes. Moreover, we tried to shed light into the mechanism through which etv6 regulates primitive erythrocytes maturation. Interestingly, we found that etv6 is a potential modulator of Notch signaling in primitive erythrocytes. Furthermore, etv6 is involved, directly or indirectly, in the repression of klf1, klf3, klf6a and klf17 genes, which are essential for primitive erythrocytes maturation. Overall, our findings suggest that the roles of pax5 and etv6 in hematopoiesis could be, at least in part, conserved in zebrafish. Interestingly, etv6 knockdown caused the alteration of the expression of some B cell markers which are impaired also by pax5 loss of function, thus, we investigated if the contemporary pax5 and etv6 knock-down could cooperate to alter the expression of these genes. However, the coinjection of pax5 and etv6 morpholinos did not cause significant alteration of igh and cd79a, thus excluding a possible cooperation between pax5 and etv6 in the regulation of their expression. Finally, we transiently expressed PAX5/ETV6 in zebrafish embryos and, despite it did not caused the deregulation of B cell markers expression, this aberrant gene is able to activate the same pathway which is altered in vitro, inducing the phosphorylation of Lck and Stat5 proteins as well as the upregulation of Stat5 effectors, suggesting that zebrafish could be a suitable model to study BCP-ALL related to PAX5/ETV6 fusion gene.