Generation and validation of molecular tools used to transgenically inhibit or overexpress miR-137. (a) Transgene used to ubiquitously express anti-miR137 sponges (named βactin:mCherry:10 × SP137). (b) Transgene used to overexpress synthetic-miR137 (named UAS:YFPs:miR137). (c) Validation of synthetic-miR137 and anti-miR137 sponge transgenic expression/activity in 3 dpf zebrafish. Transgenic animals βactin:mCherry:10 × SP137, which ubiquitously express mCherry:10 × SP137, were injected with two plasmids simultaneously (i) 503UNC:Gal4 expressing Gal4 specifically in muscle cells and (ii) UAS:YFPs:miR137 expressing YFP fused to synthetic-miR137. Muscle-specific expression of synthetic-miR137 induced by presence of Gal4 can be tracked by the presence of YFP (processed in green in the present pictures), and correlates with downregulation of mCherry fluorescent intensity, confirming efficient activity of both anti-miR137 sponges and synthetic-miR137. Mosaic expression of YFPs:miR137 was observed as the transgenes were injected and thus not stably integrated into the genome. (d) Confocal images (0.86-µm section) showing endogenous miR-137 translational repression activity on a transcript carrying anti-miR137 sponges in 3-dpf zebrafish. The injected zebrafish (βactin:mCherry:10 × SP137; SEN:GFP) expressed mCherry:10 × SP137 ubiquitously and GFP specifically in sensory neurons (Rohon–Beard cells presented in c. These neurons also expressed endogenous miR-137 (Supplementary Figures 1 and 5). Due to the presence of miR-137, mCherry:10 × SP137 transcript translation was repressed, resulting in poor mCherry expression. Compared with MO-control, injection of MO137-02 (8 ng) dramatically increased fluorescent intensity, confirming that 8 ng MO137-02 was sufficient to inhibit endogenous miR-137 activity (quantification are presented in Supplementary Figure 5). GFP, green fluorescent protein; YFP, yellow fluorescent protein.
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