Zebrafish Mll Depletion Model Phenocopies Mammalian Mll Depletion and Implicates MLL as Master Regulator of Novel Developmental Hematopoietic Targets.

Infant leukemia is an embryonal hematopoietic cancer because the characteristic translocations disrupting the MLL transcription factor initiate in utero. Zebrafish afford a unique model organism in which to understand MLL-regulated targets in intact animals and at the earliest developmental timepoints not accessible in mammals. We investigated consequences of loss of mll in zebrafish embryos to mimic the microenvironment in which MLL leukemia initiates in infants. Since disruption of one allele by the translocation creates haploinsufficiency, and failed differentiation and profound multi-lineage cytopenias are features of MLL disease, we hypothesized that the zebrafish embryo mll depletion model would uncover new links of MLL to developmental control of hematopoietic targets.

Two independent splice blocking morpholino (MO) antisense oligonucleotides titrated to no effect concentrations of mismatch controls were employed to deplete mll and verify specificity. External phenotypes were monitored and whole embryos were studied by TUNEL staining and hemoglobin staining with o-dianisidine. Effects on expression of candidate hematopoietic targets were quantified in sorted GFP⁺ precursor/myeloid and GFP⁻ non-myeloid hematopoietic cells from control or mll morphant Tg(spi1:EGFP) embryos by Q-RT-PCR. Blood cell morphologic changes were visualized on cytospins. Spatio-temporal changes in hematopoietic gene expression were studied in whole embryos by WISH. Fluorescence intensity changes were measured in live Tg reporter lines with blood cell gene promoters driving expression of fluorescent protein markers. Q-RT-PCR analysis of RNAs from unsorted Mll^+/+, Mll^+/− and Mll^−/− murine embryoid bodies (EBs) was used for validation.

Head and jaw defects in both morphants phenocopied the neuronal and craniofacial defects of Mll^−/− mice; small eyes, the eye defect of mice deficient for the Mll cofactor Meis1; and small size and delayed development, the small size of Taspase1^−/− mice from cell cycle impairments when Mll is not cleaved by Taspase1. Increased apoptosis and decreased hemoglobin staining phenocopied other features of Mll deficient mice. Depleting mll also increased or decreased hoxa9a expression in GFP⁺ or GFP⁻ cells from Tg(spi1:EGFP), respectively, and increased ccna and ccne cyclin gene expression in the GFP⁺ fraction. Moreover, deregulated gene expression in both fractions identified new mll-regulated hematopoietic targets. In the GFP⁺ precursor/myeloid fraction, lmo2, scl, cmyb and ikaros were overexpressed, and mll depletion accelerated myeloid (spi1, mpx, lcp1) gene expression. Paradoxically, an entire erythroid program (gata1, hbae1, slc4a1, alas2, sptb, epo, epoR) was upregulated in the myeloid fraction, while erythroid gene downregulation in the GFP⁻ (erythroid) cells would explain the anemia from mll depletion. Conversely, myeloid (runx1, mpx) genes were overexpressed in the GFP⁻ cells. lmo2 and ikaros expression were also increased in this fraction. These gene expression changes manifest as profound blood cell dysmorphologies. WISH showed that mll depletion in wild type embryos increased gata1 and lmo2 expression. Increases in DsRed fluorescence in Tg(gata1:DsRed) and Tg(lmo2:DsRed) mll morphant embryos further substantiated direct or indirect mll regulation of gata1 and lmo2 promoters. Q-RT-PCR demonstrated precocious Gata1 expression in Mll deficient murine EBs relative to their wild type counterparts.

The external phenotype, increased apoptosis and anemia in the morphants are critical evidence that zebrafish mll depletion phenocopies depletion of mammalian Mll and interacting factors. Prior murine models demonstrated that Mll maintains Hox expression; this work adds new information that mll has differential effects on hoxa9a in myeloid and non-myeloid cells. These studies lead to the conclusion that zebrafish mll orchestrates profound multi-lineage functions as a master regulator in the developmental control of the hematopoietic system, and has cell-type specific effects on newfound stem cell and myeloid targets and an entire erythroid program. The Gata1 expression patterns in Mll deficient murine EBs validate that Mll also directly or indirectly regulates erythropoiesis in mammals. Zebrafish are a relevant in vivo model to identify MLL-dependent pathways in blood cell development.

No relevant conflicts of interest to declare.