Figure 3. miR-9 inhibited astrogliogenesis via targeting three components of Jak-Stat pathway.

(A) Upper right panels: schematic representation of in vitro delivery of miR-9 constructs into cortical progenitors by electroporation. Lower left panels showed examples of astrocyte marker Gfap+ or neuronal marker Map2+ cells. Right panel showed overexpression of miR-9 significantly reduced the number of astrocytes 10 days after electroporation, whereas knockdown of miR-9 dramatically promoted astrogliogenesis (**p < 0.01, *p < 0.5, Mann–Whitney test). Overexpression of miR-9 in NPCs did not increase the number of neurons. Knockdown of miR-9 reduced the number of Map2+ neurons. Map2, Microtubule-Associated Protein 2. (B) Transfection of mouse NPCs with exogenous miR-9 duplex blocked phosphorylation of Stat1/3 without altering their protein levels. (C) Luciferase activity of Lifr-beta, Il6st (gp130), and Jak1 3′ UTR luciferase reporter in the presence of different combinations of control (con), miR-9, miR-9 inhibitor con, and miR-9 inhibitor in mouse NPCs. (D) miR-9 inhibited protein levels of Jak-Stat signaling components in NPCs transfected with control or miR-9 duplex. Right panels: western blotting densitometry analysis of protein level changes. Actb (β-actin) serves as the loading control. (E) A constitutively active form of Stat3, Stat3C bypassed the effect of miR-9 inhibition on astrocyte differentiation. (F) Schematic representation of Ngn1-regulated miR-9 signaling that modulates Stat1/3 phosphorylation to control cell fate specification. Ngn1 up-regulates miR-9 expression during neurogenesis. miR-9 reduces protein levels of Lifr-beta, Il6st, and Jak1 of Jak-Stat signaling pathway by targeting their 3′ UTRs, which in turn abolish Stat1/3 phosphorylation to suppress astrogliogenesis. Crebbp: CREB binding protein, Smad1: Mothers Against DPP Homolog 1 (Drosophila), Ep300: E1A Binding Protein p300, E-protein: ubiquitous basic-Helix-Loop-Helix proteins, such as E12 or E47. The dotted arrows show the inhibition regulations.

DOI: http://dx.doi.org/10.7554/eLife.06885.006

Figure 3—figure supplement 1. miR-9 and Ngn1 inhibited astrogliogenesis.

(A) Transfection of mouse E11 cortical NPCs with exogenous miR-9 duplex significantly reduced the number of Gfap+ astrocytes in vitro. (B) miR-9 suppressed activation of glial-specific Gfap promoter, with little effect on the neurogenic promoter Neurod1. (C) Ngn1 promoted neurogenesis and suppresses astrogliogenesis. Western blotting analysis showed that overexpression of Ngn1 in mouse E11 cortical NPC promoted Tuj1 expression, while reduced Gfap+ expression (left panels). Tuj1, beta III tubulin, a neuronal marker. Right panel showed overexpression of Ngn1 in mouse E11 cortical NPC promoted neurogenesis (Tuj1+ cells).

DOI: http://dx.doi.org/10.7554/eLife.06885.007

Figure 3—figure supplement 2. miR-9 targeted Jak-Stat signaling pathway.

(A) Predicted duplex formation between mouse Lifr-beta, Il6st, and Jak1 3′ UTR (top) and miR-9 (bottom). (B) Luciferase activity of wild type Lifr-beta 3′ UTR and miR-9 binding region mutant Lifr-beta 3′ UTR reporter genes with or without miR-9 duplex in mouse E11 cortical NPCs (*p < 0.0001, Wilcoxon-Mann-Whitney test). (C, D) Ngn1 suppressed the expression of three major components of Jak-Stat signaling pathway. (C) Western blot analysis of Ngn1 suppressed the protein levels of Lifr-beta, Il6st, and Jak1. Actb served as the loading control. (D) Overexpression of Ngn1 decreased the activity of luciferase reporter fused to 3′ UTRs of Il6st, Lifr-beta, and Jak1 mRNAs. (*p < 0.05, **p < 0.005, Wilcoxon-Mann-Whitney test).

DOI: http://dx.doi.org/10.7554/eLife.06885.008