The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development

Abstract

Neuronal gene expression is tightly regulated in developing CNS. Here, we demonstrate the anti-neural function of phosphatase SCP1 (small C-terminal domain phosphatase 1) during development. We further show that the neuron-enriched microRNA miR-124 directly targets SCP1-3' untranslated region (UTR) to suppress SCP1 expression. In developing spinal cord, expression of miR-124 and SCP1 is complementary, and miR-124 antagonism phenocopies SCP1 overexpression and vice versa. In P19 cells, miR-124 suppresses SCP1 expression and induces neurogenesis, and SCP1 counteracts this proneural activity of miR-124. Our results suggest that, during CNS development, timely down-regulation of SCP1 is critical for inducing neurogenesis, and miR-124 contributes to this process at least in part by down-regulating SCP1 expression.

Figure 1.

SCP1 as an anti-neural factor in the chick neural tube. (A) In situ hybridization on chick embryos reveals that SCP1 is widely expressed but down-regulated in the lateral neuronal zone. (B) SCP1-misexpressing cells (GFP⁺ cells) in the lateral zone lack the NF expression. (C) Misexpression of SCP1 leads to ectopic BrdU⁺-proliferating cells in the lateral zone (cells in red circle), and this is markedly enhanced by coexpression of REST. (D,E) While ectopic BrdU⁺ cells lack post-mitotic marker p27^kip1 (arrows in D), they maintain expression of progenitor gene Pax6 (arrows in E). (F,G) Phosphatase-inactive SCP1 mutant SCP1-pi induces ectopic and precocious NF⁺ neuronal differentiation (arrows in F) and expression of p27^kip1 (arrows in G) in the medial zone of the chick neural tube.

Figure 2.

SCP1-3′UTR as a direct target of miR-124. (A,B) In situ detection of miR-124 in chick and mouse embryos shows that miR-124 is highly and specifically expressed in the lateral neuronal zone of the neural tube and DRG, complementary to the expression pattern of SCP1. (C) Three evolutionarily conserved miR-124 target sites are found at the 3′UTR of SCP1 mRNA. Seed match sequences are marked in red. (D) Luciferase assays with the GL3∷SCP1-3′UTR reporter in HEK293 cells. Both synthetic miR-124 RNA duplexes (miR-124) and miR-124 expression vector (CMV-124) repress luciferase expression from GL3∷SCP1-3′UTR, while the miR-124 mutant and miR-128 have no effect. GL3 vector alone is minimally affected by miRNAs. The B-segment of SCP1-3′UTR containing the second miR-124 target site is suppressed by miR-124, and mutation on the miR-124 target site in the B-segment (Bm) abolishes the repression by miR-124. (E) Electroporation of GFP∷SCP1-3′UTR directs a high level of GFP expression in the medial zone and a lower GFP expression in the NF⁺ lateral zone of the chick neural tube. Coelectroporation of miR-124 diminishes GFP expression from GFP∷SCP1-3′UTR, whereas miR-124-mt has no effect. CMV-LacZ was included as an electroporation indicator.

Figure 3.

Proneural activity of miR-124 in the chick neural tube. (A–D) Overexpression of miR-124 in the chick spinal cord. (A) Electroporation of miR-124 RNA duplexes (with CMV-GFP as an electroporation indicator) increases miR-124 expression in the developing chick spinal cord 2 d post-electroporation as detected with in situ hybridization. (B–D) miR-124 overexpression triggers ectopic neurogenesis (p27^kip1+, NF⁺ post-mitotic neuronal cells in B,C) in the more medial area of the neural tube, accompanied by compromised progenitor cell proliferation (D). (E–H) Inhibition of miR-124 function in the chick spinal cord. Electroporation of 2′-OMe antisense miR124 (anti-miR-124) with CMV-GFP interferes with miR-124 detection by in situ hybridization (E), decreases the expression of neuronal markers p27^kip1 and NeuN in the lateral zone of the neural tube (F,G), and simultaneously triggers laterally positioned BrdU⁺-proliferating cells (cells in red circle in H).

Figure 4.

miR-124-induced neurogenesis is antagonized by SCP1 in P19 mouse embryonic cells. (A) Western analyses show that, in P19 cells, miR-124 expression induces post-mitotic cell marker p27^kip1 and neuronal marker TuJ, while suppressing proliferation marker cyclinA. Notably, transfection indicator GFP expression is lower in miR-124-transfected cells. (B) RT–PCR assays reveal that expression of miR-124, but not miR-124-mt, reduces SCP1 and induces proneural bHLH factors Ngn2 and NeuroD and neuronal marker TuJ. Quantitative RT–PCR results are as shown. (C) Neuronal differentiation analyses in transfected P19 cells. miR-124 expression triggers TuJ⁺ neuronal differentiation, which is compromised by cotransfection of the miR-124-resistant SCP1 vector but not by the miR-124-sensitive SCP1 vector containing SCP1-3′UTR (SCP1∷ 3′UTR). Percentage of neuronal differentiation is measured by percentage of TuJ⁺ neurons among GFP⁺-transfected cells. (D) Negative feedback loop between the anti-neural REST/SCP1 and proneural miR-124 pathways contributes to a rapid and efficient transition of cellular phenotypes between neural progenitors and post-mitotic neurons (see text). Blue and red denote the regulatory pathways in nonneurons and neurons, respectively.