EENA promotes myeloid proliferation through stimulating ERK1/2 phosphorylation in zebrafish

The EEN (extra eleven nineteen) gene is one of fusion partners of MLL (mixed-lineage leukemia), located on chromosome 19p13. Here, we cloned two een genes (designated as eena and eenb) in zebrafish, which are assigned to linkage groups 8 and 2, respectively. Whole-mount in situ hybridization assay showed that eena and eenb have overlapping, but distinct expression patterns during embryogenesis. Ubiquitious or targeted overexpression of eena, but not eenb, into wild-type or transgenic embryos (green-fluorescent protein-labeled myeloid progenitors) induced a significant proliferation and ectopic distribution of myeloid progenitors in the yolk sac. Using a morpholino-antisense gene knockdown approach, we showed that the number of myeloid progenitors and their downstream mature myelomonocytic cells was significantly decreased in the eena-deficient embryos. Mechanistically, overexpression of eena selectively stimulated ERK phosphorylation and increased the level of transcription factor c-Fos in vitro and in vivo, while eena lacking the SH3 domain completely abolished these effects. Furthermore, a MAPK/ERK kinase (MEK) inhibitor, PD98059, blocked the eena-induced cell proliferation and activation of ERK signaling. The results suggest that eena plays an important role in the development of myeloid cell through activation of the ERK pathway and may provide a valuable reference for future studies of the role of EEN in leukemogenesis.