Emi1 Is Required for Normal Cell Cycle Progression in Zebrafish Myelopoiesis and Likely Functions as a Haploinsufficient Tumor Suppressor on Chromosome 6q in Human Leukmias.

Myelopoiesis is a developmental process through which stem cells give rise to mature monocytes and granulocytes by means of a tightly regulated cellular program. The molecular mechanisms controlling myelopoiesis are incompletely understood, as are the abnormalities in this process that give rise to myeloid cell diseases, such as leukemia, myelodysplasia, and myeloproliferative syndromes. These diseases are commonly associated with somatically acquired large chromosomal deletions, making the identification of the causative tumor suppressor genes difficult. To learn more about the genes required for normal myelopoiesis, which we postulated might be mutated during the molecular pathogenesis of myeloid malignancies, we performed a screen using a panel of insertional mutants provided by Nancy Hopkins (MIT) to identify zebrafish lines with deficiencies in myeloperoxidase (mpo), a gene specifically expressed in granulocytes. As part of this screen, we identified the line hi2648, in which homozygous mutants have a severe reduction in the numbers of mature myeloid cells expressing either mpo or l-plastin, and in which the small numbers of residual myeloid cells are abnormally large. We bred hi2648 mutant fish to a line carrying the pu.1:GFP transgene and found that the homozygous mutant embryos displayed decreased numbers but relatively large GFP-positive cells, which were also observed in cytospins of FACS-purified, GFP-expressing cells, consistent with our in situ analysis. Hi2648 harbors a retroviral insertion that disrupts the gene emi1 between exons one and two, resulting in decreased levels of emi1 RNA assayed by whole- mount in situ analysis. We confirmed that the hematopoietic phenotype in this line is due to disruption of emi1 by showing that we could phenocopy the mutant using emi1-specific morpholinos and also by rescuing the mutant phenotype using forced expression of emi1 RNA. The Emi1 gene product has been shown to inhibit anaphase-promoting complex during interphase of the cell cycle. We analyzed emi1-mutant embryos for cell cycle defects by FACS analysis of propidium-iodide stained cells at 21 hpf, and showed that impaired emi1 expression results in an accumulation of cells in G2 phase, presumably due to premature activation of APC during S- and G2-phase with an inability to accumulate adequate levels cyclin B. We also observed a range of increased of levels of cellular DNA content greater than the normal 4n complement, suggesting that loss of emi1 also promotes abortive endoreduplication. Mutational analysis indicated gene-specific disruption in of emi1 in a human leukemia cell line, suggesting that this gene represents one of the long-sought haploinsufficient tumor suppressors located on the long arm of human chromosome 6 (del 6q), whose loss promotes genomic instability during leukemogenesis and the progression of MDS to AML in man.