During vertebrate development, the initial wave of hematopoiesis produces cells that help to shape the developing circulatory system and oxygenate the early embryo. The differentiation of primitive erythroid and myeloid cells occurs within a short transitory period, and is subject to precise molecular regulation by a hierarchical cascade of transcription factors. The TALE-class homeodomain transcription factors Meis and Pbx function to regulate embryonic hematopoiesis, but it is not known where Meis and Pbx proteins participate in the hematopoietic transcription factor cascade. To address these questions, we have ablated Meis1 and Pbx proteins in zebrafish, and characterized their molecular effects on known markers of primitive hematopoiesis. Embryos lacking Meis1 and Pbx exhibit a severe reduction in the expression of gata1, the earliest marker of erythroid cell fate, and fail to produce visible circulating blood cells. Concomitant with a loss of gata1, Meis1- and Pbx-depleted embryos exhibit downregulated embryonic hemoglobin (hbae3) expression, and possess increased numbers of pu.1-positive myeloid cells. gata1-overexpression rescues hbae3 expression in Pbx-depleted; meis1-morphant embryos, placing Pbx and Meis1 upstream of gata1 in the erythropoietic transcription factor hierarchy. Our study conclusively demonstrates that Meis1 and Pbx act to specify the erythropoietic cell lineage and inhibit myelopoiesis.

Summary NUP98‐HOXA9 [t(7;11) (p15;p15)] is associated with inferior prognosis in de novo and treatment‐related acute myeloid leukaemia (AML) and contributes to blast crisis in chronic myeloid leukaemia (CML). We have engineered an inducible transgenic zebrafish harbouring human NUP98‐HOXA9 under the zebrafish spi1(pu.1) promoter. NUP98‐HOXA9 perturbed zebrafish embryonic haematopoiesis, with upregulated spi1expression at the expense of gata1a. Markers associated with more differentiated myeloid cells, lcp1, lyz, and mpx were also elevated, but to a lesser extent than spi1, suggesting differentiation of early myeloid progenitors may be impaired by NUP98‐HOXA9. Following irradiation, NUP98‐HOXA9‐expressing embryos showed increased numbers of cells in G2‐M transition compared to controls and absence of a normal apoptotic response, which may result from an upregulation of bcl2. These data suggest NUP98‐HOXA9‐induced oncogenesis may result from a combination of defects in haematopoiesis and an aberrant response to DNA damage. Importantly, 23% of adult NUP98‐HOXA9‐transgenic fish developed a myeloproliferative neoplasm (MPN) at 19–23 months of age. In summary, we have identified an embryonic haematopoietic phenotype in a transgenic zebrafish line that subsequently develops MPN. This tool provides a unique opportunity for high‐throughput in vivo chemical modifier screens to identify novel therapeutic agents in high risk AML.

Prodigiosenes, possessing a 4-methoxypyrrolyldipyrrin skeleton, are known for their anti-cancer activity. Structural modification of the C-ring resulted in a series of prodigiosenes that displayed promising activity against leukemia cell lines during in vitro analysis against the NCI 60 cancer cell line panel. Further in vivo studies of these compounds using the zebrafish model showed persistence of anti-leukemia properties in human K562 chronic myelogenous leukemia cells.