Qiu R , Liu K , Liu Y , Mo W , Flynt AS , Patton JG , Kar A , Wu JY , He R .

R01 GM075790 NIGMS NIH HHS

	Fig. 1. Expression pattern of miR-124a during the development of Xenopus laevis. (A–G) Expression of miR-124a (dark blue) as detected by whole mount in situ hybridization in Xenopus embryos at stage 18 (A), stage 23 (B), stage 33 (C), stage 37 (D), stage 46 (E), and dissected adult retinas (F) and brain (G), shown from the anterior (A), lateral (B–D), or dorsal views (E and G). (H–L) Restricted location of miR-124a in the eye (H–K) and brain (L) is shown on transversal (H–K) or coronal sections (L) at stage 33 (H), stage 37 (I), stage 40 (J), and stage 46 (K and L). The pink arrow indicates the most peripheral part of the dorsal ciliary marginal zone. Scale bars: 300 μm (A–E); 1 mm (F and G); 100 μm (H–L). Ce, cerebellum; Die, diencephalon; Mes, mesencephalon; OB, olfactory bulb; Rho, rhobemcephalon; Tel, telencephalon.
	Fig. 2. Down-regulation of miR-124a shows no obvious effect on the morphogenesis of the Xenopus eye. Embryos were unilaterally injected with 0.2 pmol 2′-O-methyl antisense oligonucleotides against miR-124a (A–H) or a control miRNA, miR-198 (I). (A–C) The eyes/head of an injected Xenopus embryo at stage 46 shown from the dorsal (A) or lateral (B and C) view. (D and E) The expression of miR-124a (dark blue) detected by whole mount in situ hybridization at stage 46 (D) and stage 33 (E). The eyes/heads of embryos are shown from the dorsal view. (F–H) The structure of the eyes of an injected embryo at stage 46 shown in a transversal section (5 μm) stained with hematoxylin (blue)–eosin solution (pink). (G) and (H) show the eyes in (F) under a higher magnification. (I) Expression of miR-124a (dark blue) was not affected by the microinjection of miR-198 inhibitor at stage 33. Scale bars: 300 μm (A–E, I); 100 μm (F); 50 μm (G and H). The yellow dashed line indicates the midline of the embryos separating the uninjected control side (C) and the injected side (Inj). The injected side is traced by the light blue staining of beta-galactosidase co-injected with miR-124a or miR-198. L, lens; NR, neural retina; RPE, retina pigmented epithelium.
	Fig. 3. Effects of miR-124a overexpression on eye development in Xenopus. miR-124a or control (miR-198) miRNA precursors (0.025 pmol) were microinjected into one Xenopus dorsal-animal blastomere at the 8-cell stage. (A) The head region of the injected Xenopus embryo grown to stage 46 is shown from the dorsal or lateral view. The pink arrow marks an enlarged and pigmented optic stalk-like structure induced by miR-124a injection. (B) The structure of wild-type and abnormal eyes of a stage 46 embryo injected by miR-124a is shown on a series of transverse sections (5 μm) from anterior to posterior. The sections were stained with hematoxylin (blue)–eosin solution (pink). Pink arrows indicate the optic nerve. The red arrowhead shows the reduced retina ganglion layers. (C) The expression (dark blue) of Pax2, Rx1, and Pax6 after miR-124a overexpression was detected by whole mount in situ hybridization at stages 13, 20, and 33. Stage 13 and 20 embryos are shown from the anterior view, and the eyes of stage 33 embryos are shown from the lateral view. Scale bars: 300 μm (A and C); 100 μm (B).
	Fig. 4. miR-124a and its candidate targets. (A) An evolutionarily conserved miR-124 target site is found at the 3′ UTR of Lhx2 mRNAs in different species. Seed sequences are marked in red. Homologous sites are marked by asterisks. (B) Expression of the predicted targets of miR-124a: Lhx2, Hairy2, Gli3, NeuroD1 and Otx2 were detected by whole mount in situ hybridization. Embryos were injected with 0.05 pmol miR-124a precursor molecules into one dorsal-animal blastomere at the 8-cell stage and collected at stage 13 (anterior view), stage 20 (anterior view) or stage 33 (lateral view and dorsal view). The pink arrowhead indicates the reduced expression of Lhx2 on the injected side. Scale bars: 500 μm. (C) Luciferase assays were carried out in HEK293 cells using the pCS2-Luc-3′ UTR reporters of Lhx2, Gli3, and the positive (anti-miR-124a) and negative (Pax6) controls. Luciferase activity after transfection of the miR-124a precursor (pre-124a) or control precursor (pre-control) was normalized by that of the control cells without precursor transfection. Data are expressed as the means of three independent transfections ±SD, each carried out in triplicate. Asterisks indicate where the miR-124a transfected group was significantly different from the control precursor group (P < 0.05). (D) In situ hybridization detection of miR-124a and Lhx2 expression in Xenopus embryos at stage13 (anterior view), stage 20 (anterior view) and stage 33 (transection at the eye-level). Scale bars: 300 μm (stage 13, stage 20); 100 μm (stage 33).
	Fig. 5. miR-124a overexpression inhibits cell proliferation during retinogenesis. miR-124a or control miRNA precursor (0.025 pmol) was co-injected with GFP mRNA into one dorsal-animal blastomere of a Xenopus embryo at the 8-cell stage, and cell proliferation was detected by BrdU incorporation. (A and B) Transversal section of a stage 33 embryo at the level of the eye showing that the number of proliferating cells (BrdU-positive, red) on the miR-124a overexpressed side (miR-124a) was much lower than that on the control side (C). All cell nuclei were counterstained with Hoechst 33258 (blue). Scale bar: 100 μm. (C) The ratio of proliferating retinal cells (BrdU-positive/Hoechst-labeled cells) in either the injected side (inj) or the control side (C) of embryos injected with miR-124a (pre-miR-124a) or control (pre-control) precursors. Data are expressed as means ± SE from twelve sections of four embryos. A significant difference (**P < 0.01 by one-way ANOVA followed by Duncan’s test) between the mean ratios between the injected and control sides was only detected for pre-miR-124a injected embryos.

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