miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex

Neurogenesis during the development of the mammalian cerebral cortex involves a switch of neural stem and progenitor cells from proliferation to differentiation. To explore the possible role of microRNAs (miRNAs) in this process, we conditionally ablated Dicer in the developing mouse neocortex using Emx1-Cre, which is specifically expressed in the dorsal telencephalon as early as embryonic day (E) 9.5. Dicer ablation in neuroepithelial cells, which are the primary neural stem and progenitor cells, and in the neurons derived from them, was evident from E10.5 onwards, as ascertained by the depletion of the normally abundant miRNAs miR-9 and miR-124. Dicer ablation resulted in massive hypotrophy of the postnatal cortex and death of the mice shortly after weaning. Analysis of the cytoarchitecture of the Dicer-ablated cortex revealed a marked reduction in radial thickness starting at E13.5, and defective cortical layering postnatally. Whereas the former was due to neuronal apoptosis starting at E12.5, which was the earliest detectable phenotype, the latter reflected dramatic impairment of neuronal differentiation. Remarkably, the primary target cells of Dicer ablation, the neuroepithelial cells, and the neurogenic progenitors derived from them, were unaffected by miRNA depletion with regard to cell cycle progression, cell division, differentiation and viability during the early stage of neurogenesis, and only underwent apoptosis starting at E14.5. Our results support the emerging concept that progenitors are less dependent on miRNAs than their differentiated progeny, and raise interesting perspectives as to the expansion of somatic stem cells.     

Stop the dicing in hematopoiesis: What have we learned?

MicroRNAs (miRNAs) belong to an abundant class of highly conserved small (22nt) non-coding RNAs. MiRNA profiling studies indicate that their expression is highly cell type-dependent. DICER1 is an essential RNase III endoribonuclease for miRNA processing. Hematopoietic cell type- and developmental stage-specific Dicer1 deletion models show that miRNAs are essential regulators of cellular survival, differentiation and function. For instance, miRNA deficiency in hematopoietic stem cells and progenitors of different origins results in decreased cell survival, dramatic developmental aberrations or dysfunctions in mice. We recently found that homozygous Dicer1 deletion in myeloid-committed progenitors results in an aberrant expression of stem cell genes and induces a regained self-renewal capacity. Moreover, Dicer1 deletion causes a block in macrophage development and myeloid dysplasia, a cellular condition that may be considered as a preleukemic state. However, Dicer1-null cells do not develop leukemia in mice, indicating that depletion of miRNAs is not enough for tumorigenesis. Surprisingly, we found that heterozygous Dicer1 deletion in myeloid-committed progenitors, but not Dicer1 knockout, collaborates with p53 deletion in leukemic progression and results in various types of leukemia. Our data indicate that Dicer1 is a haploinsufficient tumorsuppressor in hematopoietic neoplasms, which is consistent with the observed downregulation of miRNA expression in human leukemia samples. Here, we review the various hematopoietic specific Dicer1 deletion mouse models and the phenotypes observed within the different hematopoietic lineages and cell developmental stages. Finally, we discuss the role for DICER1 in mouse and human malignant hematopoiesis.     

Stop the dicing in hematopoiesis: What have we learned?

MicroRNAs (miRNAs) belong to an abundant class of highly conserved small (22nt) non-coding RNAs. MiRNA profiling studies indicate that their expression is highly cell type-dependent. DICER1 is an essential RNase III endoribonuclease for miRNA processing. Hematopoietic cell type- and developmental stage-specific Dicer1 deletion models show that miRNAs are essential regulators of cellular survival, differentiation and function. For instance, miRNA deficiency in hematopoietic stem cells and progenitors of different origins results in decreased cell survival, dramatic developmental aberrations or dysfunctions in mice. We recently found that homozygous Dicer1 deletion in myeloid-committed progenitors results in an aberrant expression of stem cell genes and induces a regained self-renewal capacity. Moreover, Dicer1 deletion causes a block in macrophage development and myeloid dysplasia, a cellular condition that may be considered as a preleukemic state. However, Dicer1-null cells do not develop leukemia in mice, indicating that depletion of miRNAs is not enough for tumorigenesis. Surprisingly, we found that heterozygous Dicer1 deletion in myeloid-committed progenitors, but not Dicer1 knockout, collaborates with p53 deletion in leukemic progression and results in various types of leukemia. Our data indicate that Dicer1 is a haploinsufficient tumorsuppressor in hematopoietic neoplasms, which is consistent with the observed downregulation of miRNA expression in human leukemia samples. Here, we review the various hematopoietic specific Dicer1 deletion mouse models and the phenotypes observed within the different hematopoietic lineages and cell developmental stages. Finally, we discuss the role for DICER1 in mouse and human malignant hematopoiesis.     

Dicer is required for the maintenance of notch signaling and gliogenic competence during mouse retinal development

MicroRNAs (miRNAs) are 19-25 nucleotide RNAs that regulate messenger RNA translation and stability. Recently, we performed a conditional knockout (CKO) of the miRNA-processing enzyme Dicer during mouse retinal development and showed an essential role for miRNAs in the transition of retinal progenitors from an early to a late competence state (Georgi and Reh [2010]: J Neurosci 30:4048-4061). Notably, Dicer CKO progenitors failed to express Ascl1 and generated ganglion cells beyond their normal competence window. Because Ascl1 regulates multiple Notch signaling components, we hypothesized that Notch signaling is downregulated in Dicer CKO retinas. We show here that Notch signaling is severely reduced in Dicer CKO retinas, but that retinal progenitors still retain a low level of Notch signaling. By increasing Notch signaling in Dicer CKO progenitors through constitutive expression of the Notch intracellular domain (NICD), we show that transgenic rescue of Notch signaling has little effect on the competence of retinal progenitors or the enhanced generation of ganglion cells, suggesting that loss of Notch signaling is not a major determinant of these phenotypes. Nevertheless, transgenic NICD expression restored horizontal cells, suggesting an interaction between miRNAs and Notch signaling in the development of this cell type. Furthermore, while NICD overexpression leads to robust glial induction in control retinas, NICD overexpression was insufficient to drive Dicer-null retinal progenitors to a glial fate. Surprisingly, the presence of transgenic NICD expression did not prevent the differentiation of some types of retinal neurons, suggesting that Notch inactivation is not an absolute requirement for the initial stages of neuronal differentiation.     

Deletion of Dicer in somatic cells of the female reproductive tract causes sterility

Dicer is an evolutionarily conserved ribonuclease III that is necessary for microRNA (miRNA) processing and the synthesis of small interfering RNAs from long double-stranded RNA. Although it has been shown that Dicer plays important roles in the mammalian germline and early embryogenesis, the functions of Dicer-dependent pathways in the somatic cells of the female reproductive tract are unknown. Using a transgenic line in which Cre recombinase is driven by the anti-Müllerian hormone receptor type 2 promoter, we conditionally inactivated Dicer1 in the mesenchyme of the developing Müllerian ducts and postnatally in ovarian granulosa cells and mesenchyme-derived cells of the oviducts and uterus. Deletion of Dicer in these cell types results in female sterility and multiple reproductive defects including decreased ovulation rates, compromised oocyte and embryo integrity, prominent bilateral paratubal (oviductal) cysts, and shorter uterine horns. The paratubal cysts act as a reservoir for spermatozoa and oocytes and prevent embryos from transiting the oviductal isthmus and passing the uterotubal junction to enter the uterus for implantation. Deep sequencing of small RNAs in oviduct revealed down-regulation of specific miRNAs in Dicer conditional knockout females compared with wild type. The majority of these differentially expressed miRNAs are predicted to regulate genes important for Müllerian duct differentiation and mesenchyme-derived structures, and several of these putative target genes were significantly up-regulated upon conditional deletion of Dicer1. Thus, our findings reveal diverse and critical roles for Dicer and its miRNA products in the development and function of the female reproductive tract.     

An essential role for Dicer in adipocyte differentiation

Dicer is a cellular enzyme required for the processing of pre‐miRNA molecules into mature miRNA, and Dicer and miRNA biogenesis have been found to play important roles in a variety of physiologic processes. Recently, reports of alterations in miRNA expression levels in cultured pre‐adipogenic cell lines during differentiation and findings of differences between the miRNA expression signatures of white and brown adipose have suggested that miRNA molecules might regulate adipocyte differentiation and the formation of adipose tissue. However, direct evidence that miRNAs regulate adipogenesis is lacking. To determine if Dicer and mature miRNA govern adipocyte differentiation, we utilized primary cells isolated from mice bearing Dicer‐conditional alleles to study adipogenesis in the presence or absence of miRNA biogenesis. Our results reveal that Dicer is required for adipogenic differentiation of mouse embryonic fibroblasts and primary cultures of pre‐adipocytes. Furthermore, the requirement for Dicer in adipocyte differentiation is not due to miRNA‐mediated alterations in cell proliferation, as deletion of the Ink4a locus and the prevention of premature cellular senescence normally induced in primary cells upon Dicer ablation fails to rescue adipogenic differentiation in fibroblasts and pre‐adipocytes. J. Cell. Biochem. 110: 812–816, 2010. © 2010 Wiley‐Liss, Inc.     

Timing specific requirement of microRNA function is essential for embryonic and postnatal hippocampal development

The adult hippocampus consists of the dentate gyrus (DG) and the CA1, CA2 and CA3 regions and is essential for learning and memory functions. During embryonic development, hippocampal neurons are derived from hippocampal neuroepithelial cells and dentate granular progenitors. The molecular mechanisms that control hippocampal progenitor proliferation and differentiation are not well understood. Here we show that noncoding microRNAs (miRNAs) are essential for early hippocampal development in mice. Conditionally ablating the RNAase III enzyme Dicer at different embryonic time points utilizing three Cre mouse lines causes abnormal hippocampal morphology and affects the number of hippocampal progenitors due to altered proliferation and increased apoptosis. Lack of miRNAs at earlier stages causes early differentiation of hippocampal neurons, in particular in the CA1 and DG regions. Lack of miRNAs at a later stage specifically affects neuronal production in the CA3 region. Our results reveal a timing requirement of miRNAs for the formation of specific hippocampal regions, with the CA1 and DG developmentally hindered by an early loss of miRNAs and the CA3 region to a late loss of miRNAs. Collectively, our studies indicate the importance of the Dicer-mediated miRNA pathway in hippocampal development and functions.     

Dicer is required for survival of differentiating neural crest cells

The neural crest (NC) lineage gives rise to a wide array of cell types ranging from neurons and glia of the peripheral nervous system to skeletal elements of the head. The mechanisms regulating NC differentiation into such a large number of cell types remain largely unknown. MicroRNAs (miRNAs) play key roles in regulating developmental events suggesting they may also play a role during NC differentiation. To determine what roles miRNAs play in differentiation of NC-derived tissues, we deleted the miRNA processing gene Dicer in NC cells using the Wnt1-Cre deleter line. We show that deletion of Dicer soon after NC cells have formed does not affect their migration and colonization of their targets in the embryo. However, the post-migratory NC is dependent on Dicer for survival. In the head, loss of Dicer leads to a loss of NC-derived craniofacial bones while in the trunk, cells of the enteric, sensory and sympathetic nervous systems are lost during development. We found that loss of Dicer does not prevent the initial differentiation of NC but as development progresses, NC derivatives are lost due to apoptotic cell death. When Dicer is deleted, both Caspase-dependent and -independent apoptotic pathways are activated in the sensory ganglia but only the Caspase-dependent apoptotic program was activated in the sympathetic nervous system showing that the specific endogenous apoptotic programs are turned on by loss of Dicer. Our results show that Dicer and miRNAs, are required for survival of NC-derived tissues by preventing apoptosis during differentiation.     

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19(Arf) and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19(Arf)-p53 signaling, and induces senescence in primary cells.     

MicroRNA-432 contributes to dopamine cocktail and retinoic acid induced differentiation of human neuroblastoma cells by targeting NESTIN and RCOR1 genes

MicroRNA (miRNA) regulates expression of protein coding genes and has been implicated in diverse cellular processes including neuronal differentiation, cell growth and death. To identify the role of miRNA in neuronal differentiation, SH-SY5Y and IMR-32 cells were treated with dopamine cocktail and retinoic acid to induce differentiation. Detection of miRNAs in differentiated cells revealed that expression of many miRNAs was altered significantly. Among the altered miRNAs, human brain expressed miR-432 induced neurite projections, arrested cells in G0–G1, reduced cell proliferation and could significantly repress NESTIN/NES, RCOR1/COREST and MECP2. Our results reveal that miR-432 regulate neuronal differentiation of human neuroblastoma cells.     

Comparison of miRNA profiling during airway epithelial repair in undifferentiated and differentiated cells in vitro

Respiratory epithelium is a highly integrated structure that efficiently protects lungs from extrinsic irritants thanks to rapid repair of the wound. The repair is a complex process that requires coordinated expression of networks of genes. Plausible regulators of this process are microRNAs. We investigated whether global miRNA silencing influences the epithelial repair, and whether changes in miRNA expression profile during repair are similar between two bronchial epithelial cell cultures: differentiated and undifferentiated cells. Two bronchial cell types were used:16HBE14o- and NHBE. Transfection was performed with siRNAs against Drosha and Dicer. For miRNA profiling, non-transfected cells were cultured until confluent and harvested for RNA isolation at baseline (cells before wounding) and at different time post-wounding (8, 16, 24, and 48 h). MicroRNA expression profiling was performed using TaqMan Array Human MicroRNA Card A. Target prediction was done in miRNA body map, and pathway analysis using DAVID. Cells with downregulated Drosha and Dicer demonstrated a significantly delayed wound repair in comparison to control in both cell lines. MiRNA expression profiling revealed that ten miRNAs exhibited significant changes over time after cell injury. These genes showed a similar expression pattern in both cell lines. The predicted targets of these miRNAs were then clustered by pathway analysis into six biological groups related to wound repair. Silencing of global miRNA expression confirmed that miRNAs are crucial for airway epithelial repair. Moreover, epithelial cells of two different origins demonstrated some similarities in miRNA expression pattern during wound repair, independent of differentiation state.     

MicroRNA-deficient NK cells exhibit decreased survival but enhanced function

NK cells are innate immune lymphocytes important for early host defense against infectious pathogens and malignant transformation. MicroRNAs (miRNAs) are small RNA molecules that regulate a wide variety of cellular processes, typically by specific complementary targeting of the 3'UTR of mRNAs. The Dicer1 gene encodes a conserved enzyme essential for miRNA processing, and Dicer1 deficiency leads to a global defect in miRNA biogenesis. In this study, we report a mouse model of lymphocyte-restricted Dicer1 disruption to evaluate the role of Dicer1-dependent miRNAs in the development and function of NK cells. As expected, Dicer1-deficient NK cells had decreased total miRNA content. Furthermore, miRNA-deficient NK cells exhibited reduced survival and impaired maturation defined by cell surface phenotypic markers. However, Dicer1-deficient NK cells exhibited enhanced degranulation and IFN-γ production in vitro in response to cytokines, tumor target cells, and activating NK cell receptor ligation. Moreover, a similar phenotype of increased IFN-γ was evident during acute MCMV infection in vivo. miRs-15a/15b/16 were identified as abundant miRNAs in NK cells that directly target the murine IFN-γ 3'UTR, thereby providing a potential mechanism for enhanced IFN-γ production. These data suggest that the function of miRNAs in NK cell biology is complex, with an important role in NK cell development, survival, or homeostasis, while tempering peripheral NK cell activation. Further study of individual miRNAs in an NK cell specific fashion will provide insight into these complex miRNA regulatory effects in NK cell biology.     

When TRBP leaves Dicer at the alt‐’ER

The principle underlying miRNA silencing seems rather simple: Dicer is required for the biogenesis of endogenous miRNAs, and mature miRNAs at the RNA‐induced silencing complex, RISC, bind to targets by sequence complementary, inhibiting protein expression. However, research shows that there are many degrees of complexity to miRNA regulation. A new study by Antoniou et al 1 that is published in this issue of EMBO Reports explores an interesting neuron‐specific facet of miRNA biogenesis. We learn that in neuronal dendrites, the endoplasmic reticulum (ER) acts as a regulatory domain for the dynamic encounter of TRBP and Dicer, two proteins required for the biogenesis of miRNAs, thus affecting neuron morphogenesis.