Abstract
The t(12;21)(p13;q22) chromosomal translocation resulting in the TEL-AML1 fusion gene is the most common translocation in childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) and is found in approximately 25% of cases. The translocation arises in utero, and can be found in approximately 1% of newborn infants. However, less than 1% of these children develop leukaemia and this translocation is not observed in adult ALL. This suggests that the cell of origin resulting in the eventual transformation to BCP-ALL is present and disrupted early during development but does not represent a potent oncoprotein. Further evidence for this is provided by animal models show that TEL-AML1 alone is necessary but not sufficient to trigger overt ALL.
To further delineate the effects of TEL-AML1 during developmental haematopoiesis we utilized a zebrafish model, Tg(ZβA:hTEL-AML1-EGFP), that expresses human TEL-AML1 (hTEL-AML1) under the control of zebrafish β-actin gene promoter. Previous studies have shown that approximately 2% of these fish develop B-cell leukemia with a long latency.
Primitive erythropoiesis was studied using whole mount in situ hybridisation (WISH) in Tg(ZβA:EGFP-hTEL-AML1+/-) and siblings. The expression pattern of ikarosand gata1a showed that primitive erythroid cells develop normally in Tg(ZβA:EGFP-hTEL-AML1+/-). The development of erythroid cells at later stages was studied using o-dianisidine staining, to assess the haemoglobinization of erythrocytes. Our results suggest normal differentiation of erythroblasts into erythrocytes in Tg(ZβA:EGFP-hTEL-AML1+/-) embryos and normal haemoglobinization of erythrocytes.
We next examined myelopoiesis in Tg(ZβA:EGFP-hTEL-AML1)using WISH for cebpα and lcp1, and Sudan Black (SB) staining to label mature granulocytes. Primitive myelopoiesis was unperturbed by hTEL-AML1 at 18hpf, however we observed an increased number of SB positive cells commencing at 2 days post fertilization (dpf). SB positive cells continued to increase in number until 4dpf when the difference became less pronounced and was gone by 7dpf (Figure 1). SB positive myeloid cells at these stages are most likely to be derived from the earliest definitive hematopoietic precursors, such as the transient bi-potent erythro-myeloid progenitor (EMP) because primitive myeloid cells are predominantly macrophage-like cells that do not express SB. By 4dpf when definitive hematopoietic stem and progenitor cell (HSPC) derived myelopoiesis is dominant, the observed difference in mature myeloid cell numbers has resolved.
The earliest lymphoid cells in zebrafish embryos are thymocytes. Thymocytes were visualized using WISH for gata3 (GATA binding protein 3), lck (T-cell specific tyrosine kinase) and rag1 (recombination activating gene 1). Expression of all three markers was unpertubed indicating that early T cell development is not affected by hTEL-AML1 fusion protein expression.
B lymphoid cells have not been convincingly observed in zebrafish until 3 weeks post fertilization despite evidence of VDJ rearrangements in whole embryos from 4dpf. We attempted to identify evidence of B lymphoid cells using WISH for pax5 and cd79b, however we were not able to identify any cells before 10dpf. This supports the observation that B cells do not arise until later in development, or are too low in expression level or few in number to identify by WISH.
In order to more closely recapitulate aspects of TEL-AML1 leukemia in humans, where one the remaining endogenouscopy of TEL is frequently lost at diagnosis of BCP-ALL, we have generated a knockout of zebrafish etv6 using CRISPR/Cas9. 4 Etv6 guides targeting zebrafish etv6 were more than 90% efficient at cleaving their DNA target. F0 animals with this mutational burden survived to adulthood with no increase in mortality. By contrast F0 Tg(ZβA:EGFP-hTEL-AML1+/-) injected with etv6 crispr had an increased mortality and showed a variety of developmental abnormalities. Assessment of their haematopoietic compartment is ongoing.
In summary we show that early myelopoiesis is disrupted during development in a zebrafish transgenic model expressing human TELAML1, and that this occurs prior to the stage at which definitive pluripotent HSPC-derived myelopoiesis is dominant. This supports the hypothesis that effects of TELAML1 during developmental haematopoiesis underpin its oncogenic potential during childhood.
No relevant conflicts of interest to declare.
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