Fig. 2

Canonical Wnt signaling is downregulated in fto morphants zebrafish.

(A) Dual luciferase assay using the β-catenin responsive TopFlash construct shows loss of reporter assay activity in ftoMO embryos analysed at both 24 hpf (con: 1.000 SEM ±0.073, ftoMO: 0.491 SEM ±0.105) and 48 hpf (con:1.000 SEM ±0.103, ftoMO 0.580 SEM ±0.066) stages. (B) Lef1 transcripts, a canonical Wnt target gene, were analysed by in situ hybridisation (ISH) at 48 hpf. Fto morphants showed marked loss of lef1 expression in the optic-tectum (arrows). Scale bar: 500 μm. n = con 56/66, ftoMO 40/53. (C) Loss of β-Catenin was confirmed in fto morphants by western blotting at 48 hpf. β-Catenin protein levels were quantified relative to the loading control (Actin). (D) ISH analysis of ctnnb1 (zebrafish β-catenin 1) at 48 hpf showed upregulation of transcripts specifically in areas of the lateral hindbrain (arrowheads). Scale bar: 500 μm. n = con 70/70, ftoMO 65/68 (E) Fto morphant Tg(7xTCF-Xla.Siam:GFP)^ia4 display loss of GFP accumulation in both the Telen- and Diencephalic regions of the brain when compared to uninjected controls at 48 hpf, embryos viewed from a dorsal perspective. Scale bar: 100 µm; n = con 20/20, ftoMO 18/20.