In adult hippocampus, new neurons are continuously generated from neural stem cells (NSCs), but the molecular mechanisms regulating adult neurogenesis remain elusive. We found that Wnt signaling, together with the removal of Sox2, triggered the expression of NeuroD1 in mice. This transcriptional regulatory mechanism was dependent on a DNA element containing overlapping Sox2 and T-cell factor/lymphoid enhancer factor (TCF/LEF)-binding sites (Sox/LEF) in the promoter. Notably, Sox/LEF sites were also found in long interspersed nuclear element 1 (LINE-1) elements, consistent with their critical roles in the transition of NSCs to proliferating neuronal progenitors. Our results describe a previously unknown Wnt-mediated regulatory mechanism that simultaneously coordinates activation of NeuroD1 and LINE-1, which is important for adult neurogenesis and survival of neuronal progenitors. Moreover, the discovery that LINE-1 retro-elements embedded inCorrespondence should be addressed to T.K. (t.warashina@aist.go.jp). 8 Present address: Cell Biology Research Center, Genome Research Laboratories, Wako Pure Chemical Industries, Ltd., Amagasaki, Hyogo, Japan. 9 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Neuroscience website. AUTHOR CONTRIBUTIONS NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript the mammalian genome can function as bi-directional promoters suggests that Sox/LEF regulatory sites may represent a general mechanism, at least in part, for relaying environmental signals to other nearby loci to promote adult hippocampal neurogenesis.In the neurogenic niche of the adult mammalian brain, self-renewing NSCs give rise to committed neuronal progenitors in the subgranular zone (SGZ) of the dentate gyrus 1 .Astrocytes are an essential cell population that defines the SGZ niche and astrocyte-derived factors have instructive effects to promote adult neurogenesis 2,3 . Recently, it has been shown that Wnt3 expression persists in the adult hippocampus and Wnt3 is released by astrocytes to regulate adult neurogenesis in vitro and in vivo 4 . In the canonical Wnt/β-catenin pathway, the TCF transcription factor transduces Wnt/β-catenin signals to activate downstream target genes 4-9 . However, the target genes of Wnt/β-catenin signaling that are responsible for promoting adult neurogenesis have not been identified. Moreover, the regulatory mechanism underlying Wnt-mediated neuronal differentiation has not yet been elucidated.NeuroD1 is a proneural basic helix-loop-helix (bHLH) transcription factor that is essential for the development of the CNS, particularly for the generation of granule cells in the hippocampus and cerebellum 10,11 . Environmental signals regulate adult neurogenesis, at least in part, through the activation of NeuroD1 (refs. 12,13 Here, we found that the transcriptional activation of NeuroD1 is dependent on canonical Wnt/ β-catenin activation and removal of Sox2 repression from the Neurod1 promoter in a sequencespeci...

We have constructed an allosterically controllable novel enzyme (designated maxizyme) that can be transcribed in vivo under the control of a human tRNA(Val) promoter. The maxizyme has sensor arms that can recognize target sequences, and in the presence of such a target sequence only, it can form a cavity that can capture catalytically indispensable Mg2+ ions. As a target for a demonstration of the potential utility of the maxizyme, we chose BCR-ABL mRNA, the translated products of which cause chronic myelogenous leukemia. Only the maxizyme (but not conventional ribozymes) had extremely high specificity and high-level activity, not only in vitro but also in cultured cells including BV173 cells derived from a patient with a Philadelphia chromosome. The maxizyme induced apoptosis only in leukemic cells with this chromosome.

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The cleavage of RNA can be accelerated by a number of factors. These factors include an acidic group (Lewis acid) or a basic group that aids in the deprotonation of the attacking nucleophile, in effect enhancing the nucleophilicity of the nucleophile; an acidic group that can neutralize and stabilize the leaving group; and any environment that can stabilize the pentavalent species that is either a transition state or a short-lived intermediate. The catalytic properties of ribozymes are due to factors that are derived from the complicated and specific structure of the ribozyme-substrate complex. It was postulated initially that nature had adopted a rather narrowly defined mechanism for the cleavage of RNA. However, recent findings have clearly demonstrated the diversity of the mechanisms of ribozyme-catalyzed reactions. Such mechanisms include the metal-independent cleavage that occurs in reactions catalyzed by hairpin ribozymes and the general double-metal-ion mechanism of catalysis in reactions catalyzed by the Tetrahymena group I ribozyme. Furthermore, the architecture of the complex between the substrate and the hepatitis delta virus ribozyme allows perturbation of the pK(a) of ring nitrogens of cytosine and adenine. The resultant perturbed ring nitrogens appear to be directly involved in acid/base catalysis. Moreover, while high concentrations of monovalent metal ions or polyamines can facilitate cleavage by hammerhead ribozymes, divalent metal ions are the most effective acid/base catalysts under physiological conditions.

In the present study, we demonstrated that insulin is produced not only in the mammalian pancreas but also in adult neuronal cells derived from the hippocampus and olfactory bulb (OB). Paracrine Wnt3 plays an essential role in promoting the active expression of insulin in both hippocampal and OB-derived neural stem cells. Our analysis indicated that the balance between Wnt3, which triggers the expression of insulin via NeuroD1, and IGFBP-4, which inhibits the original Wnt3 action, is regulated depending on diabetic (DB) status. We also show that adult neural progenitors derived from DB animals retain the ability to give rise to insulin-producing cells and that grafting neuronal progenitors into the pancreas of DB animals reduces glucose levels. This study provides an example of a simple and direct use of adult stem cells from one organ to another, without introducing additional inductive genes.

Nineteen different functional RNAs were synthesized for an investigation of the actions of ribozymes, in vitro and in vivo, under the control of two different promoters, tRNA or U6, which localize transcripts either in the cytoplasm or in the nucleus. No relationships were found between the activities of these RNAs in cultured cells and the kinetic parameters of their respective chemical cleavage reactions in vitro, indicating that in no case was chemical cleavage the rate-limiting step in vivo. For example, a hepatitis delta virus (HDV) ribozyme, whose activity in vitro was almost 3 orders of magnitude lower than that of a hammerhead ribozyme, still exhibited similar activity in cells when an appropriate expression system was used. As expected, external guide sequences, the actions of which depend on nuclear RNase P, were more active in the nucleus. Analysis of data obtained with cultured cells clearly demonstrated that the cytoplasmic ribozymes were significantly more active than the nuclear ribozymes, suggesting that mature mRNAs in the cytoplasm might be more accessible to antisense molecules than are pre-mRNAs in the nucleus. Our findings should be useful for the future design of intracellularly active functional molecules.Since the discovery of the first two ribozymes (1, 2), several new types of ribozyme with self-cleavage activity have been found in nature (3-8). Small ribozymes that can be designed to cleave RNA strands intermolecularly include hammerhead, hairpin, and HDV 1 ribozymes. These trans-acting ribozymes recognize their RNA substrates via formation of Watson-Crick base pairs, and they cleave these RNAs in a sequence-specific manner. Because of their specificity, trans-acting ribozymes show promise as tools for the dysfunction of target RNAs (9 -21).The constitutive expression of a ribozyme in vivo, under the control of a strong promoter, represents an attractive strategy for the application of trans-acting ribozymes to gene therapy. As described in our previous reports (22, 23), we have succeeded in establishing an effective ribozyme expression system, with subsequent efficient transport of transcripts to the cytoplasm, which is based on a promoter that is recognized by RNA polymerase III (pol III). High levels of expression under the control of the pol III promoter are advantageous for the exploitation of ribozymes in vivo. Therefore, we chose an expression system with the promoter of a human gene for tRNA Val . Many ribozymes, such as hammerheads and hairpins, have been effectively expressed under the control of promoters of gene for tRNAs (9, 11-15, 20, 21, 24).A major advantage of our tRNA Val -directed expression system is that, with appropriate modification of the tRNA Val portion, it is possible to colocalize the expressed ribozyme in the cytoplasm with its target mRNA (14,15,22,23,25). Ribozymes expressed under the control of the tRNA Val promoter are exported to the cytoplasm as effectively as natural tRNAs via the action of Xpo(t), 2 a tRNA-binding protein (26, 27) that functions wi...

It has been demonstrated that shortened forms of (stem II-deleted) hammerhead ribozymes with low intrinsic activity form very active dimers with a common stem II (very active short ribozymes capable of forming dimers were designated maxizymes). Intracellular activities of heterodimeric maxizymes and conventional ribozymes, under the control of a human tRNA Val -promoter, were compared against the cleavage of HIV-1 tat mRNA. The pol III-driven maxizymes formed very active heterodimers, and they successfully cleaved HIV-1 tat mRNA in mammalian cells at two sites simultaneously. The cleaved fragments were identified directly by Northern blotting analysis. Despite the initial concerns that a complicated dimerization process and formation of inactive homodimers were involved in addition to the process of association with the target, the overall intracellular activities of tRNA Val -driven maxizymes were significantly higher in mammalian cells than those of two sets of independent, conventional hammerhead ribozymes that were targeted at the same two sites within HIV-1 tat mRNA. Because the tRNA Val -driven maxizymes tested to date have been more effective than tRNA Val -driven ''standard'' hammerhead ribozymes, the tRNA Val -driven heterodimeric maxizymes appear to have potential utility as gene-inactivating agents.

Formation of the primitive streak (PS) is the initial specification step that generates all the mesodermal and endodermal tissue lineages during early differentiation. Thus, a therapeutically compatible and efficient method for differentiation of the PS is crucial for regenerative medicine. In this study, we developed chemically defined serum-free culture conditions for the differentiation of embryonic stem (ES) cells into the PS-like cells. Cultures supplemented with Wnt showed induction of expression of a PS marker, the brachyury gene, followed by induction of the anterior PS markers goosecoid and foxa2, a posterior PS marker, evx1, and the endoderm marker sox17. Similar differentiation of PS by Wnt was also observed in human ES cells. Moreover, we revealed that the activation of the Wnt canonical pathway is essential for PS differentiation in mouse ES cells. These results demonstrated that Wnt is an essential and sufficient factor for the induction of the PS-like cells in vitro. These conditions of induction could constitute the initial step in generating therapeutically useful cells of the definitive endoderm lineage, such as hepatocytes and pancreatic endocrine cells, under chemically defined conditions.