Liu K et al. (2011), MiR-124 regulates early neurogenesis in the opt...

XB-ART-42435

Nucleic Acids Res 2011 Apr 01;397:2869-79. doi: 10.1093/nar/gkq904.

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MiR-124 regulates early neurogenesis in the optic vesicle and forebrain, targeting NeuroD1.

Liu K , Liu Y , Mo W , Qiu R , Wang X , Wu JY , He R .

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MicroRNAs (miRNAs) are involved in the fine control of cell proliferation and differentiation during the development of the nervous system. MiR-124, a neural specific miRNA, is expressed from the beginning of eye development in Xenopus, and has been shown to repress cell proliferation in the optic cup, however, its role at earlier developmental stages is unclear. Here, we show that this miRNA exerts a different role in cell proliferation at the optic vesicle stage, the stage which precedes optic cup formation. We show that miR-124 is both necessary and sufficient to promote cell proliferation and repress neurogenesis at the optic vesicle stage, playing an anti-neural role. Loss of miR-124 upregulates expression of neural markers NCAM, N-tubulin while gain of miR-124 downregulates these genes. Furthermore, miR-124 interacts with a conserved miR-124 binding site in the 3'-UTR of NeuroD1 and negatively regulates expression of the proneural marker NeuroD1, a bHLH transcription factor for neuronal differentiation. The miR-124-induced effect on cell proliferation can be antagonized by NeuroD1. These results reveal a novel regulatory role of miR-124 in neural development and uncover a previously unknown interaction between NeuroD1 and miR-124.

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Species referenced: Xenopus
Genes referenced: ctrl elavl3 lhx2 mmut ncam1 neurod1 odc1 pax6 tdrd6 tubb2b twist1

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	Figure 1. MiR-124 regulates cell proliferation and neurogenesis in the optic vesicle and forebrain. (A) Proliferating cells were detected with a BrdU (red) incorporation assay. Hoechst (blue) was applied to label the nuclei of all cells. The dashed line in the schematic diagram [images from Nieuwkoop and Faber, 1994, Normal Table of Xenopus laevis (Daudin)] indicates the location of the transverse sections in the developing eye. In the optic vesicle (arrow) and forebrain (arrow head) of embryos at stage (st.) 22/23, the BrdU-positive cell ratio was significantly reduced when a miR-124 inhibitor (Anti-124) was injected, but significantly increased when an miR-124 precursor (Pre-124) was applied. In the optic cup (st.33/34), injection of either control inhibitor (Anti-ctrl) or precursor (Pre-ctrl) molecules gave no significant change in cell proliferation compared with the uninjected control (Uninj). The bar graph illustrates the BrdU-positive ratio of the transverse sections (mean ± SEM, 24 sections from six embryos). Scale bar: 100 μm. (B) Expression of NCAM and N-tubulin are significantly upregulated with downregulation of miR-124, but significantly downregulated with overexpression of miR-124. ODC and –RT are the internal and negative controls, respectively, for the RT–PCR procedure. The bar graph illustrates the gene expression level analyzed by real-time RT–PCR. Means ± SEM are from three independent experiments. The values of injected groups were compared with those of uninjected controls by one-way ANOVA followed by the Duncan test. P and #P < 0.05; *P and ##P < 0.01.
	Figure 2. Downregulation of miR-124 increases the expression of NeuroD1 in the optic vesicle. (A) Expression of NeuroD1 increased in stages 20 and 22/23 embryos but not in stages 33/34 embryos injected (Inj.) with Anti-124, as indicated by in situ hybridization, while expression of Lhx2 was not affected at any of these stages. Yellow dashed lines indicate the midlines of Stage 20 embryos. Red dots circle the position of the optic vesicle/cup. Scale bar: 500 μm. (B) RT–PCR and real-time PCR quantification confirm that expression of NeuroD1, but not Lhx2, in stage 22/23 embryos is upregulated on loss of miR-124. (C) Correspondingly, the expression of erC increased and that of xtwi decreased. P and #P < 0.05; *P and ##P < 0.01.
	Figure 3. MiR-124 overexpression reduces the expression of NeuroD1. RT–PCR (gel panel) and real-time PCR (bar graph) conditions are the same as those in Figure 2. (A) Gain of miR-124 resulted in downregulation of both NeuroD1and Lhx2 at Stages 22/23. (B) Expression of elrC decreased and that of xtwi increased. P and #P < 0.05; *P and ##P < 0.01.
	Figure 4. NeuroD1 antagonizes miR-124-induced cell proliferation. Transverse sections of the optic vesicle in stage 22/23 embryos injected with Pre-124 (0.025 pmol) and/or NeuroD1 (10 pg). Proliferzating cells were immunohistochemically stained with phosphohistone-H3 (pH3) antibody (red). Nuclei were labeled with Hoechst33 258 (blue). The pH3-positive cell ratio of transverse sections was shown in the bar graph (Mean ± SEM, 16 sections from four embryos). More proliferating cells in the brain and optic vesicle were detected in the Pre-124 group compared with the uninjected blank control. Injection of NeuroD1 mRNA alone had no obvious effect on cell proliferation. Co-injection with Pre-124 plus NeuroD1 restored cell proliferation to the level of the blank control. Scale bar: 100 μm. P < 0.05; *P < 0.01.
	Figure 5. MiR-124 targets NeuroD1. (A) An evolutionarily conserved miR-124 target element (red) is located at the 3′-UTR of NeuroD1 mRNAs in humans (hsa), mice (mmu), rats (rno), Xenopus tropicalis (xtr) and Xenopus laevis (xla). The minimal free energy (Mfe) of xla-NeuroD1 pairing to xla-miR-124, xtr-miR-124 and mmu-miR-124 was below –20 kcal/mol. A mutant NeuroD1 plasmid (xla-NeuroD1-Mut) was constructed with mutations in the underlined positions which pair with the miR-124 seed sequence (2–7 nt). Homologous sites are marked by asterisks. (B) Luciferase assays were carried out in the 293T cell line using pCS2-Luc-NeuroD1 3′-UTR reporters (in xla). Positive (Anti-miR-124, Lhx2) and negative (Pax6, NeuroD1-Mut) controls were set. Anti-miR-124 almost completely blocks luciferase activity. The relative luciferase activity of the NeuroD1 group is significantly lower than that of the negative control groups and similar to that of the Lhx2 positive control. Means ± SD are from three independent experiments. P < 0.05; P < 0.01; **P < 0.001. (C) In situ hybridization of miR-124 and NeuroD1 at stage 22/23. MiR-124 is weakly expressed in the eye (arrow) in comparison with expression of NeuroD1 as shown in the lateral view. Transverse sections at the level of the eye showed that the expression patterns of miR-124 and NeuroD1 were partially complementary in the forebrain (arrow head) and optic vesicle (arrow).
	Figure 6. A putative scheme of the regulatory effect of miR-124 on NeuroD1 in early eye and brain development. NeuroD1, which represses cell proliferation and promotes neuronal differentiation, is one of the target genes of miR-124. The arrow represents promotion; the right angle represents repression.

References [+] :

Aboobaker, Drosophila microRNAs exhibit diverse spatial expression patterns during embryonic development. 2005, Pubmed

	Figure 1. MiR-124 regulates cell proliferation and neurogenesis in the optic vesicle and forebrain. (A) Proliferating cells were detected with a BrdU (red) incorporation assay. Hoechst (blue) was applied to label the nuclei of all cells. The dashed line in the schematic diagram [images from Nieuwkoop and Faber, 1994, Normal Table of Xenopus laevis (Daudin)] indicates the location of the transverse sections in the developing eye. In the optic vesicle (arrow) and forebrain (arrow head) of embryos at stage (st.) 22/23, the BrdU-positive cell ratio was significantly reduced when a miR-124 inhibitor (Anti-124) was injected, but significantly increased when an miR-124 precursor (Pre-124) was applied. In the optic cup (st.33/34), injection of either control inhibitor (Anti-ctrl) or precursor (Pre-ctrl) molecules gave no significant change in cell proliferation compared with the uninjected control (Uninj). The bar graph illustrates the BrdU-positive ratio of the transverse sections (mean ± SEM, 24 sections from six embryos). Scale bar: 100 μm. (B) Expression of NCAM and N-tubulin are significantly upregulated with downregulation of miR-124, but significantly downregulated with overexpression of miR-124. ODC and –RT are the internal and negative controls, respectively, for the RT–PCR procedure. The bar graph illustrates the gene expression level analyzed by real-time RT–PCR. Means ± SEM are from three independent experiments. The values of injected groups were compared with those of uninjected controls by one-way ANOVA followed by the Duncan test. P and #P < 0.05; *P and ##P < 0.01.
	Figure 2. Downregulation of miR-124 increases the expression of NeuroD1 in the optic vesicle. (A) Expression of NeuroD1 increased in stages 20 and 22/23 embryos but not in stages 33/34 embryos injected (Inj.) with Anti-124, as indicated by in situ hybridization, while expression of Lhx2 was not affected at any of these stages. Yellow dashed lines indicate the midlines of Stage 20 embryos. Red dots circle the position of the optic vesicle/cup. Scale bar: 500 μm. (B) RT–PCR and real-time PCR quantification confirm that expression of NeuroD1, but not Lhx2, in stage 22/23 embryos is upregulated on loss of miR-124. (C) Correspondingly, the expression of erC increased and that of xtwi decreased. P and #P < 0.05; *P and ##P < 0.01.
	Figure 3. MiR-124 overexpression reduces the expression of NeuroD1. RT–PCR (gel panel) and real-time PCR (bar graph) conditions are the same as those in Figure 2. (A) Gain of miR-124 resulted in downregulation of both NeuroD1and Lhx2 at Stages 22/23. (B) Expression of elrC decreased and that of xtwi increased. P and #P < 0.05; *P and ##P < 0.01.
	Figure 4. NeuroD1 antagonizes miR-124-induced cell proliferation. Transverse sections of the optic vesicle in stage 22/23 embryos injected with Pre-124 (0.025 pmol) and/or NeuroD1 (10 pg). Proliferzating cells were immunohistochemically stained with phosphohistone-H3 (pH3) antibody (red). Nuclei were labeled with Hoechst33 258 (blue). The pH3-positive cell ratio of transverse sections was shown in the bar graph (Mean ± SEM, 16 sections from four embryos). More proliferating cells in the brain and optic vesicle were detected in the Pre-124 group compared with the uninjected blank control. Injection of NeuroD1 mRNA alone had no obvious effect on cell proliferation. Co-injection with Pre-124 plus NeuroD1 restored cell proliferation to the level of the blank control. Scale bar: 100 μm. P < 0.05; *P < 0.01.
	Figure 5. MiR-124 targets NeuroD1. (A) An evolutionarily conserved miR-124 target element (red) is located at the 3′-UTR of NeuroD1 mRNAs in humans (hsa), mice (mmu), rats (rno), Xenopus tropicalis (xtr) and Xenopus laevis (xla). The minimal free energy (Mfe) of xla-NeuroD1 pairing to xla-miR-124, xtr-miR-124 and mmu-miR-124 was below –20 kcal/mol. A mutant NeuroD1 plasmid (xla-NeuroD1-Mut) was constructed with mutations in the underlined positions which pair with the miR-124 seed sequence (2–7 nt). Homologous sites are marked by asterisks. (B) Luciferase assays were carried out in the 293T cell line using pCS2-Luc-NeuroD1 3′-UTR reporters (in xla). Positive (Anti-miR-124, Lhx2) and negative (Pax6, NeuroD1-Mut) controls were set. Anti-miR-124 almost completely blocks luciferase activity. The relative luciferase activity of the NeuroD1 group is significantly lower than that of the negative control groups and similar to that of the Lhx2 positive control. Means ± SD are from three independent experiments. P < 0.05; P < 0.01; **P < 0.001. (C) In situ hybridization of miR-124 and NeuroD1 at stage 22/23. MiR-124 is weakly expressed in the eye (arrow) in comparison with expression of NeuroD1 as shown in the lateral view. Transverse sections at the level of the eye showed that the expression patterns of miR-124 and NeuroD1 were partially complementary in the forebrain (arrow head) and optic vesicle (arrow).
	Figure 6. A putative scheme of the regulatory effect of miR-124 on NeuroD1 in early eye and brain development. NeuroD1, which represses cell proliferation and promotes neuronal differentiation, is one of the target genes of miR-124. The arrow represents promotion; the right angle represents repression.