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Figure 1. miR-182 Is Localized in RGC Axons
(A) Heatmap representing the average expression of mature miRNAs from two axonal small RNA-sequencing (sRNA-seq) libraries prepared from stage 37/38 retinal cultures. The figure is sorted by decreasing axonal average values.
(B) Fluorescent ISH on stage 35/36 RGC GCs cultured in vitro for 24 hr.
(C) TaqMan qPCR performed on RNA extracted from laser-captured stage 37/38 RGC axons. U6 snRNA was used as positive control, because it is found in developing axons (Natera-Naranjo et al., 2010, Zhang et al., 2013 and Hancock et al., 2014).
RT−, no template negative control; snRNAU6, U6 snRNA. Scale bar, 5 μm (B). See also Figure S1 and Table S1.
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Figure 2. miR-182 Is Active and Enriched in RGC Axons
(A) Sensor construct design.
(B) Schematic representation of the experimental protocol.
(C–E) Illustrative images of RGC GCs (C), RGC soma (D), or PRs (E) following retinal electroporation of control-Sensor or miR-182-Sensor. Clear examples of dGFP/mCherry ratio decrease are shown in (C) and (E).
(F and G) Quantification of the dGFP/mCherry ratio at the RGC GCs, soma, or PRs.
Values are mean ± SEM. Mann-Whitney test (F) and two-way ANOVA followed by Tukey post hoc test (G), ∗p < 0.05, ∗∗∗∗p < 0.0001. ns, nonsignificant; CMV, cytomegalovirus promoter; CS, complementary sequence; dGFP, destabilized GFP; INL, inner nuclear layer; PRL, photoreceptor layer; RGCL, retinal ganglion cell layer. Scale bars, 20 μm (B, D, and E) and 5 μm (C). See also Figure S2.
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Figure 3. In Vivo, miR-182 Is Involved in RGC Axon Targeting but Not Long-Range Pathfinding
(A, C, and E) Schematic representation of the experimental protocols and representative images of brains, where RGC axons are stained with DiI or expressing mCherry. Arrows delineate the width of the pathway (A).
(B, D, and F) Quantification of pathway width. (B) Schematic representation of the methodology applied for pathway width measurements.
Values are mean ± SEM. Numbers of brains analyzed are between brackets. Two-way ANOVA followed by Bonferroni post-test, ∗p < 0.05, ∗∗p < 0.01. Cont, control; MO, morpholino oligomer; RGC, retinal ganglion cell. Scale bars, 150 μm (A, top panels) and 50 μm (A, bottom panel; C; and E). See also Figure S3.
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Figure 4. miR-182 Is Involved in Slit2-Driven RGC Axon Guidance and Targeting In Vivo and In Vitro
(A) Schematic representation of the experimental protocol and representative images of brains, where RGC axons are stained with DiI.
(B) Quantification of pathway width. Numbers of brains analyzed are between brackets.
(C and D) Schematic representation of the experimental protocols (C) and representative images (D) of brains, where RGC axons are stained with HRP and Slit2 mRNAs are revealed by ISH.
(E–G) In vitro turning assay on stage 35/36 RGC axons cultured for 24 hr and isolated from their cell bodies. (E) Representative images of control of miR-182 morphant RGC GC before and 60 min after being exposed to a gradient of Slit2 established from a pipette (top right corner) set at 45° angle from the initial direction of growth. (F) Tracings of RGC axons are analyzed. The source of the guidance cue is indicated by the arrowhead. Red, black, and blue traces represent, respectively, repulsive behaviors (angle < −5°), nonsignificant changes in the direction of growth (−5° < angle < 5°), and attractive turning (angle > 5°). (G) Quantification of the average turning angle. Numbers of GCs analyzed are between brackets.
Values are mean ± SEM (B and G). Two-way ANOVA followed by Bonferroni post-test (B) or Mann-Whitney test (G), ∗p < 0.05, ∗∗p < 0.01. Cont, control; HRP, horseradish peroxidase; ISH, in situ hybridization; MO, morpholino oligomer; RGC, retinal ganglion cell. Scale bars, 150 μm (A, top panels), 50 μm (A, bottom panel, and D), and 30 μm (E). See also Figures S4 and S5.
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Figure 5. miR-182 Targets Cfl1 mRNA and Regulates Its Expression in RGC Axons
(A) Top predicted miR-182 targets expressed in Xenopus laevis growth cones.
(B) Sequence alignment of the 3′ UTR of Cfl1. The predicted miR-182 binding site is highlighted in red.
(C) Schematic representation of Xenopus Cfl1-3′ UTR, subcloned downstream of a dual Renilla:Firefly luciferase reporter.
(D) Quantification of reporter activity in HEK293T cells.
(E and F) Representative images (E) and quantification (F) of Cfl1 immunostaining. White lines delineate RGC growth cones. Bath application of Slit2 was used at a suboptimal concentration to avoid collapse.
Values are mean ± SEM (D and F). Numbers of GCs analyzed are indicated in bars (F). ANOVA followed by Bonferroni post-test, ∗∗∗p < 0.001. ns, nonsignificant; cfl1, Cfl1; cont, control; MO, morpholino oligomer; MUT, mutated; WT, wild-type. Scale bar, 5 μm (E). See also Table S2.
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Figure 6. miR-182 Is Required for Slit2-Induced Local Translation of Cfl1 in RGC GCs
(A) Schematic representation of the experimental protocol. After 24 hr, RGC axons were isolated from their cell bodies. Bath application of Slit2 at a suboptimal concentration was used to avoid collapse. Vehicle was used as control. Recovery of the newly synthesized Kaede green protein was monitored over time.
(B) Quantification of the recovery of Kaede green signal. Data are presented as the percentage change of the fluorescence intensity (F) over time. Numbers of GCs analyzed are indicated in the legend of the graph.
(C and D) Representative pre- and post-photoconversion images of severed control (C) or miR-182 morphant (D) axons.
Values are mean ± SEM (B). Kruskal-Wallis test, ∗p < 0.05, ∗∗p < 0.01. Scale bars, 10 μm (C and D). Cont, control; LPS, local protein synthesis; MO, morpholino oligomer. See also Figure S6.
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Figure 7. Slit2 Inhibits miR-182 Activity in RGC Axons without Decay
(A, D, and F) Schematic representation of the experimental paradigm. Stage 35/36 retinal explants were cultured for 24 hr, and then Slit2 or vehicle were bath applied for 10 min.
(B) Illustrative images of GCs from miR-182-Sensor-electroporated RGCs grown in culture. A clear example of dGFP/mCherry ratio increase is shown in (B).
(C and E) Quantification of the dGFP/mCherry fluorescent ratio at the GC.
(G) Illustrative images of explants and axons before and after LCM.
(H) Illustrative gel of RT-PCR reaction for β-actin (β-act), MAP2, and histone H4 (H4) mRNA from cultured axons collected from stage 37/38 by LCM. In MAP2, H4, and β-act negative controls, PCR template was omitted.
(I) Quantification of miR-182 by the ΔΔCt method in LCM axons.
Values are mean ± SEM (C, E, and I). Mann-Whitney test, ∗p < 0.05. ns, nonsignificant; LCM, laser capture microdissection; RT−, RT no template negative control. Scale bars, 5 μm (B) and 200 μm (G). See also Figure S7.
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Figure S1: Validation of axonal RNA purification and sequencing, Related to Figure 1.
(A) Schematic representation of the experimental protocol. 1000 stage 37/38 eye explants were
cultured. After 48h, explants were manually removed, and total RNA was extracted from the axonal
fraction thus obtained. Small RNA cDNA libraries were generated and sequenced. (B) Representative
images of 48h cultured stage 37/38 eye explant, before (left) and after (right) removal of the eye (star)
and contaminating cells (arrowhead). Scale bar 300µm. (C) RT-PCR for MAP2 and β-actin mRNA
performed on RNA extracted from the axonal or eye fraction. Both β-actin and MAP2 mRNA are
detected in the eye fraction while only β-actin mRNA is detected in the axonal one, suggesting that the
RNA extracted from the axonal fraction after removal of the explant is devoid of RNA contamination
from cell bodies and dendrites. (D) Pearson correlation analysis of sequencing reads of axonal
miRNAs between the two replicates. Values on axes show log2(cpm+1) for each sample. The Pearson
correlation coefficient of 0.93 reported on top of the plot indicates a good correlation between the two
replicates. (E) Illustrative gel showing TaqMan qPCR performed on RNA extracted from laser
captured RGC layer detecting traces of miR-182 in RGC soma. RNA-U6 was used as a positive
control. RT-, RT no template negative control; PCR-, PCR no template negative control.
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