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. 2014 Jan 15;34(3):969–979. doi: 10.1523/JNEUROSCI.1910-13.2014

Figure 2.

Figure 2.

miRNA (miR)-132 controls axon branching of mouse RGCs. A, Dissociated retinal cultures from E15–E16 mouse embryos were electroporated with expression plasmids for miRNA-132 or a control (scrambled) miRNA, and were incubated for 3 d in the presence or absence of 10 ng/ml BDNF. The plasmids contain an RFP expression cassette for identification of electroporated cells (Pathania et al., 2012). Typical examples for RGC branching patterns are shown under conditions as indicated. Scale bar, 10 μm. B, Quantification of the number of branches per axon expressing miRNA-132 or an miRNA with a scrambled sequence, in the presence or absence of BDNF. The expression of miRNA-132 leads to an increase in the number of branches even in the absence of BDNF. C, Average axon length was not affected. D, Quantification of the number of branches per axon of retinal cultures expressing control sponge or miRNA-132 sponge in the presence or absence of BDNF. Axons, in which miRNA-132 function is abolished by expression of sponges, do not show an increase in branching in response to BDNF. E, The average axon length was not affected. B–E, Error bars denote SEM. Significance is indicated as ***p < 0.001, *p < 0.05. n.s., Not significant. The number of axons analyzed (from a total of three independent experiments) is shown at the bottom of the bars. Statistical analysis was done in GraphPad using one-way ANOVA and Tukey post hoc test. F, Characterization of the miRNA-132 sponge in chick retinal cultures. Single-cell cultures from E8 chick retinae coexpressing GFP-containing sponge plasmids containing multiple binding sites for either miRNA-132 or a mutated binding site (contr.), together with an RFP-containing sensor with only two binding sites for miRNA-132. Here RFP fluorescence relative to GFP fluorescence is given (see Materials and Methods). The miRNA-132 sponge, but not the control sponge, sequesters miRNA-132 away from the sensor, leading to a stronger RFP fluorescence. Error bars denote SEM. Significance is indicated as ***p < 0.001. A.U., Arbitrary units.