Fig. S4

Validation of miR-9 targets conserved across vertebrates (related to Figure 3)

(A, B) Whole-mount in situ hybridization against tlx (A) or ocl (B) in larvae at 72 hpf injected with the control MO, the miR-9 MO or the miR-9 RNA mimic. tlx and ocl behave like a miR-9 target showing an increase in the miR-9 knockdown and a decrease in the miR-9 gain of function. (C, D) Schematic representation of the zebrafish oc1 (C) and oc2 (D) mRNA indicating the sequence of the putative miR-9 binding sites in the 3'UTR (red). Whole-mount in situ hybridization against oc1 (C) and oc2 (D) in larvae at 72 hpf injected with the control MO, the miR-9 MO or the miR-9 RNA mimic. onecut family mRNAs behave like miR-9 targets showing an increase in the miR-9 knockdown and a decrease in the miR-9 gain of function. (E, F) Fluorescent sensor assay to test the functionality of miR-9 binding sites in the 3'UTR of oc1 (E) and ocl (F) showing that miR-9 mediates oc1 and ocl inhibition via the 3'UTR. TagRFP and EGFP protein expression in embryos at 24 hpf: embryos were co-injected with the egfp mRNA containing the 3'UTR of oc1 (n=7) or ocl (n=3) fused to the SV40pA and the internal control, tagrfp mRNA (n=8 and 3 respectively) in the presence or absence of miR-9 mimic. The ratio between the level of EGFP and TagRFP proteins fluorescence shows a decrease in EGFP expression, but not TagRFP, in the presence of the miR-9 RNA mimic. Dorsal view of the brain with anterior up. Lateral view of the retina. Scale bars: 100 μm. Error bars represent s.d. *P<0.05, **P<0.001, ***P<0.0005, determined by t-test, two-tailed.