A functional study of miR-124 in the developing neural tube

Abstract

Neural development is a highly orchestrated process that entails precise control of gene expression. Although microRNAs (miRNAs) have been implicated in fine-tuning gene networks, the roles of individual miRNAs in vertebrate neural development have not been studied in vivo. We investigated the function of the most abundant neuronal miRNA, miR-124, during spinal cord development. Neither inhibition nor overexpression of miR-124 significantly altered the acquisition of neuronal fate, suggesting that miR-124 is unlikely to act as a primary determinant of neuronal differentiation. Two endogenous targets of miR-124, laminin gamma 1 and integrin beta1, were identified, both of which are highly expressed by neural progenitors but repressed upon neuronal differentiation. Thus miR-124 appears to ensure that progenitor genes are post-transcriptionally inhibited in neurons.

Figure 1.

Inhibiting miR-124 does not affect neuronal differentiation. (A–C) miR-124 in situ hybridization (ISH) on chick embryos transfected with LNA-modified miR-124 antisense oligonucleotides (LNA-124as, 100 μM) showed decreased hybridization signals on the transfected side of the spinal cord at 20 and 45 hpe (arrows). Scrambled oligonucleotides (LNA-124scr) did not cause such a decrease. The image next to each ISH photograph shows the expression of the cotransfected GFP plasmid on an adjacent section. (D–F) Transfection of LNA-124as did not affect the expression of neuronal markers Tuj1, p27/Kip1, and NeuN at 20 hpe. Images on the left of the dashed lines show the whole neural tube sections, those on the right show only the transfected side.

Figure 2.

Overexpressing miR-124 in the chick neural tube. (A,B) The stem-loop structures of miR-124 and miR-124mt with red letters designating the sequences of mature miRNAs. Blue letters are the mutations introduced that should disrupt seed matches between miR-124 and its targets. (C) Northern blots probed for miR-124 or miR-124mt showed similar levels of expression from the overexpression plasmids in 293T cells. Lanes 1 and 4 are RNAs from untransfected 293T cells. Lanes 2, 3, 5, and 6 are RNAs from 293T cells transfected with the plasmid expressing miR-20, miR-124, miR-124, and miR-124mt, respectively. (D) miR-124 ISH showed high levels of expression (arrows) from the miR-124 plasmid in the transfected neural tube at 45 hpe. (E) The schematic drawing of a miRNA sensor plasmid. The two blue ovals represent two copies of complementary sequences to the miRNA that the sensor is designed to detect. The gray ovals represent scrambled sequences. (Pcag) Chick β-actin promoter; (d4EGFPn) nuclear-localized destabilized EGFP with a half-life of 4 h; (mRFPn) nuclear-localized monomeric red fluorescent protein. (F–H) At 45 hpe, most cells transfected with the sensor for miR-124 (pSensor-124) were yellow, expressing both GFP and RFP (F). Cotransfection of the miR-124 plasmid strongly repressed GFP expression (G), which was not observed with the miR-124mt plasmid (H).

Figure 3.

Overexpressing miR-124 does not promote overt neuronal differentiation. (A) Ectopic miR-124 did not affect the expression of the neural progenitor marker Sox2. (B) Cell cycle analysis by BrdU labeling. miR-124 was cotransfected with a plasmid expressing a nuclear localized GFP (GFPn) to facilitate cell counting. (C) Quantifications of the fraction of transfected cells (GFP⁺) that were mitotically active (BrdU⁺GFP⁺) showed no difference in embryos transfected with miR-124, miR-124mt, or the empty vector pENTR. (D–F) Overexpression of miR-124 did not increase the expression of neuronal markers Tuj1, p27/Kip1, or NeuN at 20 hpe.

Figure 4.

Ectopic miR-124 causes basal lamina defects. (A,B) Immunostaining for laminin-1 showed disintegration of basal laminae upon the overexpression of miR-124 (A, arrows). (B) The defect was located near the dorsal root entry site, shown on an adjacent section by immunostaining with BEN, an antibody that labels sensory axons (arrows). (C) The line of integrin β1 (ITGB1) that surrounds the outer surface of the neural tube was disrupted by ectopic miR-124 (arrows). (D,E) LAMC1 and ITGB1 RNAi caused similar, albeit milder, basal lamina defects (arrows). Transfected cells were marked by RFP encoded in RNAi vectors. (F) Quantifications of basal lamina defects. “Defects frequency” refers to the percentage of defective sections among all the sections examined in each embryo. (G,H) Cotransfection of an ITGB1-expressing plasmid with miR-124 partially rescued the basal lamina defect at 45 hpe (H). (G) High levels of ITGB1 expression were achieved from the transfected plasmid, with the overexpressed protein properly localized to the plasma membrane (inset).

Figure 5.

LAMC1 and ITGB1 mRNAs are endogenous targets of miR-124. (A–C) LAMC1, ITGB1, and miR-124 ISH on HH27–28 brachial-level transverse sections. (VZ) Ventricular zone; (MZ) mantle zone; (MD) motor domain; (DRG) dorsal root ganglion. (D) Luciferase assays with the luciferase gene fused to SV40, chick LAMC1, or chick ITGB1 3′-UTR. pENTR, miR-124, or miR-124mt plasmids (amounts shown in the X-axis) were cotransfected with luciferase–UTR constructs. Luciferase activities obtained from the cotransfection of pENTR and each luciferase–UTR construct were used as denominators. Cotransfection of miR-124, but not miR-124mt, specifically repressed LAMC1 and ITGB1 UTRs but not SV40 UTR. (E, panel a) During neural tube development, neural progenitors (orange) contribute to the formation and maintenance of basal laminae (BL) by producing laminin γ1 (black balls) and integrin β1 (red bars). (Panel b) Upon ectopic expression of miR-124 (short purple lines) in neural progenitors, both genes were repressed, leading to the disintegration of basal laminae. Green cells represent differentiating neurons.