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A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination.

Zhao C, Sun G, Li S, Shi Y - Nat. Struct. Mol. Biol. (2009)

Bottom Line: Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation.Moreover, TLX represses expression of the miR-9 pri-miRNA.By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Center for Gene Expression and Drug Discovery, Beckman Research Institute of City of Hope, Duarte, California, USA.

ABSTRACT
MicroRNAs have been implicated as having important roles in stem cell biology. MicroRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain and may be involved in neural stem cell self-renewal and differentiation. We showed previously that the 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 that is not prone to miR-9 regulation 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 expression of the miR-9 pri-miRNA. By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

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Overexpression of miR-9 regulates neural stem cell proliferation and differentiationa. Cell proliferation in miR-9-transfected neural stem cells as revealed by BrdU labeling (green). Merged panels show BrdU staining along with phase contrast images. A miR-9 mutant with mutations in the seed region was included as a control, with a total of 200 nM miRNAs in each transfection. b. Quantification of BrdU-positive (BrdU+) cells in miR-9-treated neural stem cells with s.d. indicated by error bars. * p=0.0008 by one-way Anova. c. Top panel is Western blot analysis of TLX expression in miR-9 transfected neural stem cells. GAPDH was included as a loading control. Lower panel is RT-PCR analysis of TLX and p21 in miR-9-transfected neural stem cells. Actin was included as a loading control. d. Overexpression of miR-9 promotes glial differentiation. Control RNA or miR-9-transfected cells were induced into differentiation for 3 days and immunostained with a GFAP-specific antibody (green). Nuclear dapi staining is shown in blue. e. Overexpression of miR-9 promotes neuronal differentiation. Control RNA or miR-9 transfected neural stem cells were induced to differentiate for 3 days and immunostained with a Tuj1-specific antibody (red). Nuclear dapi staining is shown in blue. For both sections d and e, error bars are s.d. of the mean. * p=0.03 (d), 0.018 (e) by Student's t-test. About 4,000 cells were quantified for panels d and e, respectively.
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Figure 2: Overexpression of miR-9 regulates neural stem cell proliferation and differentiationa. Cell proliferation in miR-9-transfected neural stem cells as revealed by BrdU labeling (green). Merged panels show BrdU staining along with phase contrast images. A miR-9 mutant with mutations in the seed region was included as a control, with a total of 200 nM miRNAs in each transfection. b. Quantification of BrdU-positive (BrdU+) cells in miR-9-treated neural stem cells with s.d. indicated by error bars. * p=0.0008 by one-way Anova. c. Top panel is Western blot analysis of TLX expression in miR-9 transfected neural stem cells. GAPDH was included as a loading control. Lower panel is RT-PCR analysis of TLX and p21 in miR-9-transfected neural stem cells. Actin was included as a loading control. d. Overexpression of miR-9 promotes glial differentiation. Control RNA or miR-9-transfected cells were induced into differentiation for 3 days and immunostained with a GFAP-specific antibody (green). Nuclear dapi staining is shown in blue. e. Overexpression of miR-9 promotes neuronal differentiation. Control RNA or miR-9 transfected neural stem cells were induced to differentiate for 3 days and immunostained with a Tuj1-specific antibody (red). Nuclear dapi staining is shown in blue. For both sections d and e, error bars are s.d. of the mean. * p=0.03 (d), 0.018 (e) by Student's t-test. About 4,000 cells were quantified for panels d and e, respectively.

Mentions: To examine whether miR-9 regulates neural stem cell proliferation, neural stem cells were transfected with increasing concentrations of miR-9 RNA duplexes. Cell proliferation was determined by 5-bromodeoxyuridine (BrdU) labeling of dividing cells. Transfection of miR-9 led to dose-dependent inhibition of cell proliferation (Fig. 2a, b) with a minimal effect on cell death (Supplementary Fig. 1c, d). Accordingly, reduced expression of TLX and increased expression of p21, a target gene that is repressed by TLX17, was observed in miR-9-transfected cells in a dose-dependent manner (Fig. 2c). This gene expression profile is consistent with miR-9-induced inhibition of neural stem cell proliferation (Fig. 2a, b), suggesting that miR-9 negatively regulates neural stem cell proliferation, presumably through downregulation of TLX signaling.


A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination.

Zhao C, Sun G, Li S, Shi Y - Nat. Struct. Mol. Biol. (2009)

Overexpression of miR-9 regulates neural stem cell proliferation and differentiationa. Cell proliferation in miR-9-transfected neural stem cells as revealed by BrdU labeling (green). Merged panels show BrdU staining along with phase contrast images. A miR-9 mutant with mutations in the seed region was included as a control, with a total of 200 nM miRNAs in each transfection. b. Quantification of BrdU-positive (BrdU+) cells in miR-9-treated neural stem cells with s.d. indicated by error bars. * p=0.0008 by one-way Anova. c. Top panel is Western blot analysis of TLX expression in miR-9 transfected neural stem cells. GAPDH was included as a loading control. Lower panel is RT-PCR analysis of TLX and p21 in miR-9-transfected neural stem cells. Actin was included as a loading control. d. Overexpression of miR-9 promotes glial differentiation. Control RNA or miR-9-transfected cells were induced into differentiation for 3 days and immunostained with a GFAP-specific antibody (green). Nuclear dapi staining is shown in blue. e. Overexpression of miR-9 promotes neuronal differentiation. Control RNA or miR-9 transfected neural stem cells were induced to differentiate for 3 days and immunostained with a Tuj1-specific antibody (red). Nuclear dapi staining is shown in blue. For both sections d and e, error bars are s.d. of the mean. * p=0.03 (d), 0.018 (e) by Student's t-test. About 4,000 cells were quantified for panels d and e, respectively.
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Figure 2: Overexpression of miR-9 regulates neural stem cell proliferation and differentiationa. Cell proliferation in miR-9-transfected neural stem cells as revealed by BrdU labeling (green). Merged panels show BrdU staining along with phase contrast images. A miR-9 mutant with mutations in the seed region was included as a control, with a total of 200 nM miRNAs in each transfection. b. Quantification of BrdU-positive (BrdU+) cells in miR-9-treated neural stem cells with s.d. indicated by error bars. * p=0.0008 by one-way Anova. c. Top panel is Western blot analysis of TLX expression in miR-9 transfected neural stem cells. GAPDH was included as a loading control. Lower panel is RT-PCR analysis of TLX and p21 in miR-9-transfected neural stem cells. Actin was included as a loading control. d. Overexpression of miR-9 promotes glial differentiation. Control RNA or miR-9-transfected cells were induced into differentiation for 3 days and immunostained with a GFAP-specific antibody (green). Nuclear dapi staining is shown in blue. e. Overexpression of miR-9 promotes neuronal differentiation. Control RNA or miR-9 transfected neural stem cells were induced to differentiate for 3 days and immunostained with a Tuj1-specific antibody (red). Nuclear dapi staining is shown in blue. For both sections d and e, error bars are s.d. of the mean. * p=0.03 (d), 0.018 (e) by Student's t-test. About 4,000 cells were quantified for panels d and e, respectively.
Mentions: To examine whether miR-9 regulates neural stem cell proliferation, neural stem cells were transfected with increasing concentrations of miR-9 RNA duplexes. Cell proliferation was determined by 5-bromodeoxyuridine (BrdU) labeling of dividing cells. Transfection of miR-9 led to dose-dependent inhibition of cell proliferation (Fig. 2a, b) with a minimal effect on cell death (Supplementary Fig. 1c, d). Accordingly, reduced expression of TLX and increased expression of p21, a target gene that is repressed by TLX17, was observed in miR-9-transfected cells in a dose-dependent manner (Fig. 2c). This gene expression profile is consistent with miR-9-induced inhibition of neural stem cell proliferation (Fig. 2a, b), suggesting that miR-9 negatively regulates neural stem cell proliferation, presumably through downregulation of TLX signaling.

Bottom Line: Here we show that miR-9 suppresses TLX expression to negatively regulate neural stem cell proliferation and accelerate neural differentiation.Moreover, TLX represses expression of the miR-9 pri-miRNA.By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosciences, Center for Gene Expression and Drug Discovery, Beckman Research Institute of City of Hope, Duarte, California, USA.

ABSTRACT
MicroRNAs have been implicated as having important roles in stem cell biology. MicroRNA-9 (miR-9) is expressed specifically in neurogenic areas of the brain and may be involved in neural stem cell self-renewal and differentiation. We showed previously that the 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 that is not prone to miR-9 regulation 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 expression of the miR-9 pri-miRNA. By forming a negative regulatory loop with TLX, miR-9 provides a model for controlling the balance between neural stem cell proliferation and differentiation.

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