MicroRNA-9 modulates Cajal-Retzius cell differentiation by suppressing Foxg1 expression in mouse medial pallium

Abstract

Vertebrate brain hosts a diverse collection of microRNAs, but little is known about their functions in vivo. Here we propose that mouse microRNA-9 (miR-9) targets Foxg1 mRNAs for proper generation of Cajal-Retzius cells in the medial pallium. miR-9 expression is mediolaterally graded, being most intense in the cortical hem; it contrasts with the Foxg1 expression in a reciprocal gradient. The 3' untranslated regions of tetrapod, but not of teleost, Foxg1 mRNAs conserve miR-9 target sequences and are regulated by miR-9. Gain- and loss-of-function analyses of miR-9 showed that miR-9 negatively regulates endogenous Foxg1 protein level. Moreover, miR-9 overexpression in developing telencephalon at E11.5 by electroporation resulted in ectopic Reelin-positive cells over the cortex beyond the marginal zone. In addition, inhibition of endogenous miR-9 function by antisense oligonucleotides caused the regression of Wnt3a-positive cortical hem and reduction of reelin-, p73-, and NeuroD1-positive cells.

Figure 1.

miR-9 expression in developing telencephalon. A, Whole-mount in situ hybridization at E9.0 (Aa), E9.5 (Ab), and E10.5 (Ac, Ac′). Arrows indicate the expression in preoptic area, and arrowheads indicate the expression in hindbrain. Aa–Ac, Lateral views; Ac′, a frontal view. B, Section in situ hybridization with probes at days indicated. All are frontal views. Ba, Anterior view; Bb, posterior view. Bc′–Be′, Magnified views of medial regions in Bc–Be. Arrowheads in Ba and Bb indicate the expression in medial pallium, and arrows indicate the expression in pallidum. Bc, Bc′, miR-9 expression (blue) abuts the TTR-positive (orange) choroid plexus. Bd–Be′, It is found in Wnt3a-positive cortical hem but not in the junctional epithelium (arrowheads) between the choroid plexus and cortical hem. Scale bars: A, 250 μm; Ba–Be, 200 μm; Bc′–Be′, 100 μm.

Figure 2.

miR-9 targets Foxg1 3′UTR. A, Essential roles of seed sequence and target sequence in suppression of Foxg1 3′UTR by miR-9. Effects of WT or MT miR-9-1, miR-9-2, or miR-9-3 on Luciferase expression from luciferase reporters conjugated with WT or MT Foxg1 3′UTR are shown (for the details, see supplemental material, available at www.jneurosci.org). In all the assays, the luciferase expression was nearly the same at the mRNA level. B, Foxg1 spatial expression pattern in the medial pallium. Ba, miR-9 (purple) and Foxg1 (brown) mRNA expression in E12.0 telencephalon; Bb–Bd, Foxg1 protein, Foxg1 mRNA, and Wnt3a mRNA expression boundaries. Arrows in Bb–Bd indicate the limits of cortical hem marked by Wnt3a (Bd). Scale bars: Ba, 200 μm; Bb–Bd, 100 μm. C, Conservation of miR-9 target sequences in vertebrate Foxg1 3′UTRs. D, Luciferase assay of miR-9 suppression of mouse (Mm), chick (Gg), Xenopus (Xt), and zebrafish (Dr) Foxg1 3′UTRs. E, Two-base mutations in miR-9 as indicated in red (MT1–10). Sequences assumed to make base pairing in the wild-type miR-9 with Foxg1 target sequence are boxed. F, Luciferase assay of the Foxg1-suppressing activities of miR-9-2 mutated as shown in E.

Figure 3.

Effects of miR-9 on Foxg1 protein expression in the cortex cells. A, B, The effect of miR-9-2 overexpression. A, miR-9-2WT or control vector together with SV40-EGFP was electroporated into the neocortices at E12.5, and the EGFP-positive regions were dissected out from six cerebral hemispheres at E13.5. Foxg1 expression level was examined by immunoblotting; EGFP and GAPDH3 served as loading control. Experiments were conducted two times; quantification of the bands indicated 69% and 42% reduction of Foxg1 protein by miR-9-2 WT in each experiment. B, E13.5 neocortex cells were dissociated, transfected with CMV-miR-9-2WT-SV40-EGFP or SV40-EGFP by electroporation, and cultured for 24 h. Foxg1 expression was examined by immunostaining. Scale bar, 10μm. C, The effect of miR-9-2 knockdown. miR-9AS or control oligonucleotide was electroporated with SV40-EGFP into the neocortices. Foxg1 expression level in the neocortices was examined by immunoblotting. Experiments were conducted two times; quantification of the bands indicated 2.3- and 1.8-fold increase of Foxg1 protein expression by miR-9AS in each experiment.

Figure 4.

Effects of miR-9 on Cajal–Retzius cell differentiation in vivo. A, The effect of miR-9-2 overexpression. CMV-miR-9-2WT-SV40-EGFP or SV40-EGFP was electroporated at E11.5, and the neocortices were immunostained for p73 (Aa), p21 (Ab), Tuj1 (Ac, Ac′), Reelin (Ad, Ad′), and EGFP at E13.5. Ac′, Ad′, Magnified views of the ventricular regions. B, The effect of miR-9 knockdown. miR-9AS or control oligonucleotide was electroporated with SV40-EGFP into the medial pallium, and expressions of markers indicated were examined at indicated hours after the electroporation by in situ hybridization. In each panel, the left (−) indicates the control unelectroporated side, and the right (+) indicates the electroporated side. Scale bars: A, 20 μm; B, 200 μm (scale bars in B, from top to bottom, indicate 12 and 24 h, 48 h, and 72 h, respectively).