SummaryMicroRNAs are important players in stem cell biology. Among them, microRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain. Whether miR-9 plays a role in neural stem cell self-renewal and differentiation is unknown. We showed previously that nuclear receptor TLX is an essential regulator of neural stem cell self-renewal. Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation. Introducing a TLX expression vector lacking the miR-9 recognition site rescued miR-9-induced proliferation deficiency and inhibited precocious differentiation. In utero electroporation of miR-9 in embryonic brains led to premature differentiation and outward migration of the transfected neural stem cells. Moreover, TLX represses miR-9 pri-miRNA expression. MiR-9, by forming a negative regulatory loop with TLX, establishes a model for controlling the balance between neural stem cell proliferation and differentiation.

Neural stem cell self-renewal and differentiation is orchestrated by precise control of gene expression involving nuclear receptor TLX. Let-7b, a member of the let-7 microRNA family, is expressed in mammalian brains and exhibits increased expression during neural differentiation. However, the role of let-7b in neural stem cell proliferation and differentiation remains unknown. Here we show that let-7b regulates neural stem cell proliferation and differentiation by targeting the stem cell regulator TLX and the cell cycle regulator cyclin D1. Overexpression of let-7b led to reduced neural stem cell proliferation and increased neural differentiation, whereas antisense knockdown of let-7b resulted in enhanced proliferation of neural stem cells. Moreover, in utero electroporation of let-7b to embryonic mouse brains led to reduced cell cycle progression in neural stem cells. Introducing an expression vector of Tlx or cyclin D1 that lacks the let-7b recognition site rescued let-7b-induced proliferation deficiency, suggesting that both TLX and cyclin D1 are important targets for let-7b-mediated regulation of neural stem cell proliferation. Let-7b, by targeting TLX and cyclin D1, establishes an efficient strategy to control neural stem cell proliferation and differentiation.Neural stem cells are undifferentiated precursors that retain the ability to proliferate and self-renew, and they have the capacity to give rise to both neuronal and glial lineages (1). Although the functional properties of neural stem cells have been studied extensively, molecular mechanisms underlying neural stem cell self-renewal and differentiation are only beginning to be understood. One class of factors thought to be important in this process is microRNAs (miRNAs), which are short, 20-22 nucleotide RNA molecules that are expressed in a tissue-specific and developmentally regulated manner and function as negative regulators of gene expression in a variety of eukaryotes. MiRNAs are involved in numerous cellular processes including development, proliferation, and differentiation (2, 3). Studies based on expression patterns, predicted targets, and overexpression analyses suggest that miRNAs are key regulators in stem cell biology.The lethal-7 (let-7) gene is one of the first two miRNAs discovered in Caenorhabditis elegans (C. elegans), and the first known human miRNA (4, 5). Mature let-7 is highly conserved across species in both sequence and function. It plays an important role in development and cell maturation (6-9). Let-7 is expressed in both embryonic and adult brains (10-13). Recently, increased expression and maturation of let-7 has been observed during neural cell specification (8, 9). Let-7a, one of the members of the let-7 family, has been shown to play a role in neuronal differentiation of embryonic neural progenitors (14), while let-7b has been shown to reduce the self-renewal of aging neural stem cells through targeting the high-mobility group A (HMGA) family member Hmga2 expression (15). TLX (NR2E1) is an orphan nuclear receptor that i...

The nuclear receptor TLX (also known as NR2E1) is essential for adult neural stem cell self-renewal; however, the molecular mechanisms involved remain elusive. Here we show that TLX activates the canonical Wnt/β-catenin pathway in adult mouse neural stem cells. Furthermore, we demonstrate that Wnt/β-catenin signalling is important in the proliferation and self-renewal of adult neural stem cells in the presence of epidermal growth factor and fibroblast growth factor. Wnt7a and active β-catenin promote neural stem cell self-renewal, whereas the deletion of Wnt7a or the lentiviral transduction of axin, a β-catenin inhibitor, led to decreased cell proliferation in adult neurogenic areas. Lentiviral transduction of active β-catenin led to increased numbers of type B neural stem cells in the subventricular zone of adult brains, whereas deletion of Wnt7a or TLX resulted in decreased numbers of neural stem cells retaining bromodeoxyuridine label in the adult brain. Both Wnt7a and active β-catenin significantly rescued a TLX (also known as Nr2e1) short interfering RNA-induced deficiency in neural stem cell proliferation. Lentiviral transduction of an active β-catenin increased cell proliferation in neurogenic areas of TLX-null adult brains markedly. These results strongly support the hypothesis that TLX acts through the Wnt/β-catenin pathway to regulate neural stem cell proliferation and self-renewal. Moreover, this study suggests that neural stem cells can promote their own self-renewal by secreting signalling molecules that act in an autocrine/paracrine mode. 8 Correspondence should be addressed to Y.S. (yshi@coh.org). 6,7 These authors contributed equally to this work.Note: Supplementary Information is available on the Nature Cell Biology website. AUTHOR CONTRIBUTIONSY.S. conceived and designed the study. Y.S., Q.Q., G.S., W.L., S.Y., P.Y. and C.Z. performed the experiments and analysed the data. Y.S., Q.Q., G.S., F.H.G. and R.M.E. interpreted the data. Y.S. wrote the paper with comments from Q.Q., G.S., R.T.Y., F.H.G. and R.M.E. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. The finding of neurogenesis in the adult brain led to the discovery of adult neural stem cells. Neural stem cells are defined as a subset of undifferentiated precursors that retain the ability to proliferate and self-renew and have the capacity to give rise to both neuronal and glial lineages [1][2][3][4] . Under normal conditions, neurogenesis in the adult mammalian brain is restricted to two discrete germinal centres: the subgranular layer of the hippocampal dentate gyrus 3 and the subventricular zones of the lateral ventricles 5,6 . A complete understanding of adult neural stem cells requires the identification of molecules that determine the self-renewal and multipotent characteristics of these cells.TLX is an orphan nuclear receptor that is expressed in vertebrate forebrains 7,8 . We showed previously that TLX is an important regulator of neural stem cell maintenance and self-renewal in both embryo...

Two fundamental properties of stem cells are their ability to self-renew and to differentiate. Self-renewal is an integration of proliferation control with the maintenance of an undifferentiated state. Stem cell self-renewal is regulated by the dynamic interplay between transcription factors, epigenetic control, microRNA (miRNA) regulators, and cell-extrinsic signals from the microenvironment in which stem cells reside. Recent progress in defining specific roles for cell-intrinsic factors and extrinsic factors in regulating stem cell self-renewal starts to unfold the multilayered regulatory networks. This review focuses on cell-intrinsic regulators, including orphan nuclear receptor TLX, polycomb transcriptional repressor Bmi1, high-mobility-group DNA binding protein Sox2, basic helix-loop-helix Hes genes, histone modifying enzymes and chromatin remodeling proteins, and small RNA modulators, as well as cell-extrinsic signaling molecules, such as Wnt, Notch, Sonic hedgehog (Shh), TGFalpha, EGF, and FGF. Unraveling the mechanisms by which neural stem cells renew themselves will provide insights into both basic neurosciences and clinical applications of stem cell-based cell replacement therapies for neurodegenerative diseases.

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