Fig. 3

tlx and onecut Are Direct miR-9 Targets Regulating VEGF-A Transcriptional Expression In Vivo

(A) Alignments of vertebrate TLX and ONECUT2 orthologs show strong conservation in the 3′ UTR containing a putative miR-9 binding site.

(B) Schematic representation of the TP assay. In the presence of the TP, the miR-9 binding site is not accessible and the mRNA degradation is inhibited.

(C and D) Schematic representation of the zebrafish tlx (C) and ocl (D) mRNA indicating the sequence of the putative miR-9 binding site in the 3′ UTR (red) and the alignment with miR-9 or TP. tlx and ocl mRNA behave like direct miR-9 targets, showing an increase in the TP context.

(E) Schematic representation highlighting putative Tlx (NAGTCA/purple) (Qu et al., 2010; Yu et al., 2000) and Onecut (NN(A/G)TC(A/C)A(T/G)NN/orange) (Lannoy et al., 1998; Plaisance et al., 2006) binding sites in 1.5 kb of the vegfaa promoter.

(F) Whole-mount in situ hybridization against egfp driven by 1.5 kb of the vegfaa promoter. Embryos were injected with vegfaa:egfp only, hs:tlx, or hs:ocl and were heat-shocked at 7 hpf. Embryos display ectopic expression of the egfp mRNA 2 hr after treatment, showing that Tlx and Onecut have the ability to induce the transcription via 1.5 kb of the vegfaa promoter.

(G and H) Whole-mount in situ hybridization against vegfaa (G) and huc (H) in embryos at 9 hpf. Embryos were injected with hs:tlx or hs:ocl and heat-shocked at 7 hpf. Embryos display ectopic expression of vegfaa, but not huc, 2 hr after treatment, showing that Tlx and Onecut transcription factors have the ability to induce the transcription of the endogenous vegfaa gene independently of the neuronal fate.

(I) Mosaic expression of Tlx-p2A-EGFP or Ocl-p2A-EGFP by heat shock at 8.5 hpf induces endogenous vegfaa expression in as little as 30 min in a cell-autonomous manner, suggesting a direct regulation.

(J) Whole-mount in situ hybridization against vegfaa in embryos at 48 hpf injected with the control MO, miR-9 MO, or miR-9 MO with tlx and ocl MOs. Dorsal view with anterior up. Lateral view of the retina.

Scale bars: 100 μm in (C), (D), (F)–(H), and (J) or 10 μm in (I).