Involvement of miR-9/MCPIP1 axis in PDGF-BB-mediated neurogenesis in neuronal progenitor cells

Highly conserved microRNA-9 (miR-9) has a critical role in various cellular processes including neurogenesis. However, its regulation by neurotropins that are known to mediate neurogenesis remains poorly defined. In this study, we identify platelet-derived growth factor-BB (PDGF-BB)-mediated upregulation of miR-9, which in turn downregulates its target gene monocyte chemotactic protein-induced protein 1 (MCPIP1), as a key player in modulating proliferation, neuronal differentiation as well as migration of neuronal progenitor cells (NPCs). Results indicate that miR-9-mediated NPC proliferation and neuronal differentiation involves signaling via the nuclear factor-kappa B (NF-κB) and cAMP response element-binding protein (CREB) pathways, and that NPC migration involves CREB but not the NF-κB signaling. These findings thus suggest that miR-9-mediated downregulation of MCPIP1 acts as a molecular switch regulation of neurogenesis.     

Six1 is indispensable for production of functional progenitor cells during olfactory epithelial development

The rodent olfactory epithelium (OE) is a good model system for studying the principles of stem and progenitor cell biology, because of its capacity for continuous neurogenesis throughout life and relatively well-characterized neuronal lineage. The development of mouse OE is divided into two stages, early and established neurogenesis. In established neurogenesis, which starts at embryonic day (E) 12.5, sustentacular cells and olfactory receptor neurons (ORNs) are produced from apical and basal progenitors, respectively. We previously reported that Six1(-/-) shows a lack of mature ORNs throughout development and disorganization of OE after E12.5. However, the molecular bases for these defects have not been addressed. Here, we show that Six1 is expressed in both apical and basal progenitors. In Six1(-/-) mice, apical proliferating cells were absent and no morphologically identifiable sustentacular cells were observed. Consistently, the expression of Notch2 and Jagged1 in the apical layer was absent in Six1(-/-) mice. On the other hand, basal proliferating cells were observed in Six1(-/-) animals, but the expression of Ngn1, NeuroD, Notch1, and Jagged2 in the basal layer was absent. The expression of Mash1, the determination gene for ORNs, and Hes genes was enhanced in Six1(-/-) mice. The present findings suggest that Six1 regulates production of functional apical and basal progenitors during OE development, through the regulation of various genes, such as neuronal basic helix-loop-helix (bHLH), neuronal repressor bHLH, and genes involved in the Notch signaling pathway.     

Notch signaling is required for the maintenance of enteric neural crest progenitors

Notch signaling is involved in neurogenesis, including that of the peripheral nervous system as derived from neural crest cells (NCCs). However, it remains unclear which step is regulated by this signaling. To address this question, we took advantage of the Cre-loxP system to specifically eliminate the protein O-fucosyltransferase 1 (Pofut1) gene, which is a core component of Notch signaling, in NCCs. NCC-specific Pofut1-knockout mice died within 1 day of birth, accompanied by a defect of enteric nervous system (ENS) development. These embryos showed a reduction in enteric neural crest cells (ENCCs) resulting from premature neurogenesis. We found that Sox10 expression, which is normally maintained in ENCC progenitors, was decreased in Pofut1-null ENCCs. By contrast, the number of ENCCs that expressed Mash1, a potent repressor of Sox10, was increased in the Pofut1-null mouse. Given that Mash1 is suppressed via the Notch signaling pathway, we propose a model in which ENCCs have a cell-autonomous differentiating program for neurons as reflected in the expression of Mash1, and in which Notch signaling is required for the maintenance of ENS progenitors by attenuating this cell-autonomous program via the suppression of Mash1.     

MiR-34a targeting of Notch ligand delta-like 1 impairs CD15+/CD133+ tumor-propagating cells and supports neural differentiation in medulloblastoma

Background

Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies.

Methodology/Principal Findings

In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133⁺/CD15⁺ tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1^+/- p53^-/-), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo.

Conclusions/Significance

Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.     

Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium

To clarify the mechanisms that regulate neuroendocrine differentiation of fetal lung epithelia, we have studied the expression of the mammalian homologs of achaete-scute complex (Mash1) (Ascl1 - Mouse Genome Informatics); hairy and enhancer of split1 (Hes1); and the expression of Notch/Notch-ligand system in the fetal and adult mouse lungs, and in the lungs of Mash1- or Hes1-deficient mice. Immunohistochemical studies revealed that Mash1-positive cells seemed to belong to pulmonary neuroendocrine cells (PNEC) and their precursors. In mice deficient for Mash1, no PNEC were detected. Hes1-positive cells belong to non-neuroendocrine cells. In the mice deficient in Hes1, in which Mash1 mRNA was upregulated, PNEC appeared precociously, and the number of PNEC was markedly increased. NeuroD (Neurod1 - Mouse Genome Informatics) expression in the lung was detected in the adult, and was enhanced in the fetal lungs of Hes1-null mice. Expression of Notch1, Notch2, Notch3 and Notch4 mRNAs in the mouse lung increased with age, and Notch1 mRNA was expressed in a Hes1-dependent manner. Notch1, Notch2 and Notch3 were immunohistochemically detected in non-neuroendocrine cells. Moreover, analyses of the lungs from the gene-targeted mice suggested that expression of Delta-like 1 (Dll1 - Mouse Genome Informatics) mRNA depends on Mash1. Thus, the neuroendocrine differentiation depends on basic helix-loop-helix factors, and Notch/Notch-ligand pathways may be involved in determining the cell differentiation fate in fetal airway epithelium.     

Notch1 is required for neuronal and glial differentiation in the cerebellum.

The mechanisms that guide progenitor cell fate and differentiation in the vertebrate central nervous system (CNS) are poorly understood. Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, we have addressed directly the role of Notch1 in the development of the mammalian brain. Using conditional gene ablation, we show that loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. We have also analyzed the effects of Notch1 ablation on gliogenesis in vivo. Our results show that Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium.     

Reelin sets the pace of neocortical neurogenesis

Migration of neurons during cortical development is often assumed to rely on purely post-proliferative reelin signaling. However, Notch signaling, long known to regulate neural precursor formation and maintenance, is required for the effects of reelin on neuronal migration. Here, we show that reelin gain-of-function causes a higher expression of Notch target genes in radial glia and accelerates the production of both neurons and intermediate progenitor cells. Converse alterations correlate with reelin loss-of-function, consistent with reelin controlling Notch signaling during neurogenesis. Ectopic expression of reelin in isolated clones of progenitors causes a severe reduction in neuronal differentiation. In mosaic cell cultures, reelin-primed progenitor cells respond to wild-type cells by further decreasing neuronal differentiation, consistent with an increased sensitivity to lateral inhibition. These results indicate that reelin and Notch signaling cooperate to set the pace of neocortical neurogenesis, a prerequisite for proper neuronal migration and cortical layering.     

miR-124 function during Ciona intestinalis neuronal development includes extensive interaction with the Notch signaling pathway

The nervous system-enriched microRNA miR-124 is necessary for proper nervous system development, although the mechanism remains poorly understood. Here, through a comprehensive analysis of miR-124 and its gene targets, we demonstrate that, in the chordate ascidian Ciona intestinalis, miR-124 plays an extensive role in promoting nervous system development. We discovered that feedback interaction between miR-124 and Notch signaling regulates the epidermal-peripheral nervous system (PNS) fate choice in tail midline cells. Notch signaling silences miR-124 in epidermal midline cells, whereas in PNS midline cells miR-124 silences Notch, Neuralized and all three Ciona Hairy/Enhancer-of-Split genes. Furthermore, ectopic expression of miR-124 is sufficient to convert epidermal midline cells into PNS neurons, consistent with a role in modulating Notch signaling. More broadly, genome-wide target extraction with validation using an in vivo tissue-specific sensor assay indicates that miR-124 shapes neuronal progenitor fields by downregulating non-neural genes, notably the muscle specifier Macho-1 and 50 Brachyury-regulated notochord genes, as well as several anti-neural factors including SCP1 and PTBP1. 3'UTR conservation analysis reveals that miR-124 targeting of SCP1 is likely to have arisen as a shared, derived trait in the vertebrate/tunicate ancestor and targeting of PTBP1 is conserved among bilaterians except for ecdysozoans, while extensive Notch pathway targeting appears to be Ciona specific. Altogether, our results provide a comprehensive insight into the specific mechanisms by which miR-124 promotes neuronal development.     

The notch signaling pathway is required to specify muscle progenitor cells in Drosophila.

Organization and function of the Notch signaling pathway in Drosophila are best understood with respect to its role in the process of selection of neural progenitor cells. However, there is evidence that, besides neurogenesis, the Notch signaling pathway is involved in several other developmental processes, one of which is the selection of muscle progenitor cells. Thus, the number of these cells is increased in neurogenic mutants, and it has been proposed that muscle progenitor cells are selected from clusters of equivalent cells expressing genes of the achaete-scute gene complex (AS-C). Here, I present evidence for the participation of additional elements of the Notch signaling pathway in myogenesis. Gal4 mediated expression of a Notch variant, E(spl) and Hairless shows that the selection of muscle progenitor cells obeys principles apparently identical to those acting at the selection of neural progenitor cells.     

Stem-loop binding protein is required for retinal cell proliferation,neurogenesis, and intraretinal axon pathfinding in zebrafish

In the developing retina, neurogenesis and cell differentiation are coupled with cell proliferation. However, molecular mechanisms that coordinate cell proliferation and differentiation are not fully understood. In this study, we found that retinal neurogenesis is severely delayed in the zebrafish stem-loop binding protein (slbp) mutant. SLBP binds to a stem-loop structure at the 3′-end of histone mRNAs, and regulates a replication-dependent synthesis and degradation of histone proteins. Retinal cell proliferation becomes slower in the slbp1 mutant, resulting in cessation of retinal stem cell proliferation. Although retinal stem cells cease proliferation by 2 days postfertilization (dpf) in the slbp mutant, retinal progenitor cells in the central retina continue to proliferate and generate neurons until at least 5 dpf. We found that this progenitor proliferation depends on Notch signaling, suggesting that Notch signaling maintains retinal progenitor proliferation when faced with reduced SLBP activity. Thus, SLBP is required for retinal stem cell maintenance. SLBP and Notch signaling are required for retinal progenitor cell proliferation and subsequent neurogenesis. We also show that SLBP1 is required for intraretinal axon pathfinding, probably through morphogenesis of the optic stalk, which expresses attractant cues. Taken together, these data indicate important roles of SLBP in retinal development.     

Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling

Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.