1. To study the role of TEL-AML1 in leukemogenesis in a tractable animal model that is amenable to whole-genome forward-genetic analysis and synthetic lethal screens, we established zebrafish transgenic lines expressing the TEL-AML1 fusion. The Zebrafish -actin (ZBA) and Xenopus elongation factor-1 (XEF1) promoters were used to direct TEL-AML1 expression constitutively, while the Zebrafish RAG-2 promoter (RAG2) was employed to selectively drive TEL-AML1 expression in lymphocyte precursors. Expression of TEL-AML1 led to the accumulation of immature hematopoietic progenitor cells in the kidney and spleen in the transgenic lines with constitutive expression, indicating an expansion of the progenitor cell population. A small number (1.3%) of founders and progeny of zebrafish transgenic for ZBA- or XEF1-TEL-AML1 fusion developed ALL with a latency of 8-12 months. None of the wild-type controls or RAG2-TEL-AML1 transgenic zebrafish developed leukemia. Constitutive expression of TEL-AML1 result in a premalignant state that requires additional genetic events for acquisition of leukemic. The observation that none of the RAG2 zebrafish expressing TEL-AML1 in committed lymphoid progenitors developed leukemia or progenitor expansion indicates that the leukemic stem cell associated with TEL-AML1 leukemia occurs in a cell that is more immature than the committed lymphoid progenitors where RAG2 expression is detected. The TEL-AML1 transgenic zebrafish lines provide a platform for mutagenesis screens to identify cooperating events that interact with TEL-AML-1 to induce leukemia. These fish are currently being characterized and will be used for mutagenesis studies to identify the additional genetic hits required for leukemic transformation 2. Within the preceding year we have used the zebrafish model to investigate the function of the EWSR1 gene, which is fused to the FLI-1 gene in human Ewing's sarcoma. Despite involvement of EWSR1 in Ewings and other sarcomas, the in vivo function of wild type EWSR1 remains unclear. We identified two zebrafish EWSR1 orthologues, ewsr1a and ewsr1b, and demonstrate that both genes are expressed maternally, and subsequently expressed ubiquitously throughout zebrafish embryonic development. Morpholino induced knockdown of both zebrafish ewsr1 genes led to mitotic defects with abnormal or multipolar mitotic spindles starting from the bud stage (10hpf). The abnormalities in mitotic spindles were followed by p53-mediated apoptosis in the developing central nervous system (CNS) leading to a reduction in the number of proneural cells, disorganization of neuronal networks, and embryonic lethality by 5 days post-fertilization. SiRNA silencing of EWSR1 in Hela cells resulted in mitotic defects accompanied by apoptotic cell death, indicating that the role of EWSR1 is conserved between zebrafish and human. Ewsr1 maintains mitotic integrity and proneural cell survival in early zebrafish development. 3. We have initiated studies to identify zebrafish harboring mutations in the zebrafish BRCA-2 gene. We are focusing on re-sequencing of zebrafish with germ-line mutations in BRCA-2 that were generated using chemically induced mutagenesis. Once these truncating mutations resembling those found in human patients are identified, the lines will be retrieved from the frozen semen of their parents, and lines will be established. Fish harboring the mutation will be observed for the development of ovarian cancer. This process will be expedited by crossing the mutant fish to transgenic fish with green ovaries that we have generated by fusing the green fluorescent protein to the ovarian zona pellucida promoter.
