Ablation of Dicer1 in Myeloid Progenitors Leads to Neutrophil Dysplasia and Depletion of Monocytes, Macrophages, and Myeloid Dendritic Cells

Abstract 43

MicroRNAs (miRNAs) belong to a family of small non-coding RNAs that post-transcriptionally suppresses protein-coding mRNAs. DICER1 is an RNaseIII endonuclease that is critical for processing precursor hairpin sequences into mature miRNAs. Thus, cell type- and developmental stage-specific conditional deletion of Dicer1 can be used to investigate the consequences of global miRNA depletion in myeloid cell development. We crossed mice that contained floxed Dicer1 (Dicer1^fl) alleles with different Cre-recombinase transgenic mice, in which CRE is expressed at different stages of myelopoiesis. In addition, we used a CRE-dependent YFP reporter system for the identification of cells that contain Dicer1 deleted alleles (Dicer1^δ).

Deletion of Dicer1 with VAVi-Cre transgenic mice, which is expressed in hematopoietic stem cells (HSCs), results in full depletion of hematopoiesis, indicating that Dicer1 is critical for stem cell maintenance. To investigate the role of Dicer1 in granulocyte macrophage progenitors (GMPs), we crossed CCAAT/enhancer binding protein a (C/ebpa)-Cre mice with the Dicer1 deleter strain that also harbored a conditional YFP allele. Because these mice suffered from late embryonic lethality due to pulmonal dysfunction, we performed transplantation with isolated HSCs from embryonic day 13.5 fetal livers. We observed a full deletion of Dicer1^fl alleles and a near complete depletion of miRNAs in YFP⁺ bone marrow cells. Strikingly, C/ebpa-Cre-driven deletion of Dicer1, did not affect the number of YFP⁺ multi-potent progenitors (MPPs), common myeloid progenitors (CMPs) and GMPs compared to wild type (wt) Dicer1 mice. However, complete loss of Dicer1 blocked monocytic differentiation, depleted macrophages and myelo-dendritic cells, and caused myeloid dysplasia with morphological features of Pelger-Huët anomaly. In addition, decreased colony formation in response to G-CSF and GM-CSF, and a block in myelo-dendritic cell outgrowth were observed when Dicer1^δ/δ progenitors were grown in vitro. Cytologic analysis of the Dicer1^δ/δ colonies demonstrated a strong reduced ability to produce mature macrophages and neutrophils. Instead, we observed outgrowth of blast-like cells that exhibited an increased replating capacity, which is suggestive for an enhanced self-renewal capacity.

MiRNA profiling of wt GMPs (Lin⁻Sca-1⁻c-Kit⁺CD34⁺FcgRII/III(CD16/32)^High) showed expression of 104 out of a panel of 375 well-characterized miRNAs tested. To further investigate the functions of these miRNAs, we profiled the gene expression of Dicer1^δ/δ and Dicer1^wt/δ GMPs and compared the transcriptome to wt Dicer1 control cells. In total, Dicer1 ablation resulted in 300 significantly up-regulated and 88 down-regulated transcripts in GMPs. Transcripts with predicted miRNA binding sites for miRNAs that are expressed in GMPs tended to be derepressed in the Dicer1^δ/δ cells relative to transcripts that do not have predicted miRNA binding sites (Kolmogorov-Smirnov test, p<2.2×10⁻¹⁶). Further, predicted targets of twenty miRNAs seeds including Let-7, miR-17, miR-142, miR-223, and miR-30 were significantly enriched in the up-regulated fraction of transcripts (Fisher exact test, FDR, p<0.05). Gene set enrichment analysis shows that differentially expressed genes are involved in cell death, cancer, and cellular growth and proliferation mainly. Further, we identified a set of Dicer1 controlled transcripts, which are exclusively expressed in self-renewing HSC such as Cav2 and Bach1, genes that are specific for the HSC/MPP stage, including HoxA9 and Hmga2, and genes that are erythroid progenitor specific, such as Nfe2l2 and Wwp1. In conclusion, Dicer1 ablation by C/ebpa-Cre does not affect the numbers of HSCs, CMPs, and GMPs but results in defective GMPs, which are unable to mature towards monocytes, macrophages, and myeloid dendritic cells, and give rise to a neutrophil dysplasia instead. These data thus uncover a Dicer1-controlled differentiation program in GMPs that is required for normal myelopoiesis.