XB-IMG-137867
Xenbase Image ID: 137867
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Fig. 2. fezf2 promotes Wnt/β-catenin signalling and induces neuronal differentiation through Wnt/β-catenin in vitro and in vivo. (A) fezf2 misexpression in early Xenopus embryos leads to strong dorsoanteriorisation (31/35 embryos examined showed the illustrated phenotype) compared with lacZ (β-gal) controls (39/39). (B) fezf2 misexpression enhances Smad2/3 phosphorylation and inhibits Smad1/5/8 phosphorylation as assessed in western blots. Blastula stage (st. 8) indicates the pre-activation state. Elongation factor 4E (elF4E) was used as a loading control. (C,D) qPCR shows that fezf2 promotes the expression of xnr3 (C) and sia (D) in early embryos (n=3 replicates). (E) TOPFlash assay shows that fezf2 promotes Wnt/β-catenin signalling (n=4 replicates). (F-H) fezf2 expression colocalises with active Wnt signalling in the forebrain. (F) The transgenic construct. (G) Dorsal and lateral views of stage 30 embryos; GFP signal for Wnt activity (green); Katushka signal for fezf2 expression (red); +bf, merged image with bright-field. (H) Knockdown of fezf2 reduces Wnt activity in the forebrain as assessed by expression of the 7LEF-dEGFP F1.1 Wnt reporter line. Arrowhead indicates the diencephalon. (I) The Wnt inhibitor δNTcf3 antagonises Fezf2-induced neuronal differentiation in mouse neuronal progenitors, as assessed by the induction of axonogenesis. (J) Statistics of I (n=4 replicates). (K,L) Electroporation experiments show that the Wnt inhibitor δNTcf3 antagonises Fezf2-induced neuronal differentiation in the tadpole forebrain. (K) Transverse sections of the forebrain area of stage 30 embryos electroporated correspondingly and stained for MyT1 (red), GFP (green) and with DAPI (blue). Left images, merge; right images, MyT1 alone. (L) Statistics of K (n=5 embryos). Control side is normalised to 100%. (M) qPCR analysis shows that the Wnt inhibitor δNTcf3 antagonises Fezf2-induced ngn1 expression in stage 20 animal cap explants (n=3 replicates). In all qPCR analyses, ribosomal protein L8 (rpl8) was used as an internal control. *P<0.05, **P<0.01, ***P<0.001. Error bars represent s.e.m. Scale bars: 100 µm in I; 50 µm in K. Image published in: Zhang S et al. (2014) © 2014. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license
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