XB-IMG-150862
Xenbase Image ID: 150862
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Figure S5 (related to Figure 2). fezf2 promotes Wnt/β-catenin signalling. (A) wnt8
mis-expression inhibited BMP signalling while activating TGF-β/Nodal signalling. 50
pg of either nuclear β-gal mRNA or wnt8a mRNA was injected into X. laevis embryos at 1-2 cell stage and collected at the gastrula stage (10.5) for Western blot analysis.
Phospho-Smad 2/3 antibody (TGF-β/Nodal signalling ) or phospho-Smad 1/5/8 (BMP
signalling) were used assay for the activation states of TGF-β/Nodal or BMP
signalling. A significant increase on the phosphorylation level of Smad 2/3 was
observed together with significant decrease on the phosphorylation level of Smad
1/5/8 was seen in wnt8 mRNA injected embryos. (B-G) fezf2 mis-expression
promotes Wnt-responsive dorsal markers goosecoid and chordin expression. In all
cases ribosomal protein L8 (rpl8) gene was used as internal control for qPCR analysis.
(B-D) goosecoid expression. (B) Uninjected control embryo. (C) fezf2 mRNA
overexpressing embryo. (D) qPCR analysis, showing an increase of goosecoid
expression (n=3 replicates, P<0.05). (E-G) chordin expression. (E) Uninjected control
embryo. (F) fezf2 mRNA overexpressing embryo. (G) qPCR analysis, showing an
increase of chordin expression (n=3 replicates, P<0.001). (H-M) fezf2 mis-expression
inhibits ventral markers vent1 and bmp4 expression. Embryo treatments are same as
in (B-G). (H-J) vent1 expression. (H) Uninjected control embryo. (I) fezf2 mRNA
overexpressing embryo. (J) qPCR analysis, showing a decrease of vent1 expression
(n=3 replicates, P<0.001). (K-M) bmp4 expression. (K) Uninjected control embryo.
(L) fezf2 mRNA overexpressing embryo. (M) qPCR analysis, showing a decrease in
vent1 expression (n=3 replicates, P<0.01). (N) fezf2 mis-expression leads to nuclear
accumulation of β-catenin. 250 pg of either nuclear β-gal (control) or fezf2 mRNA
was injected at 1-2 cell stage and section made at stage 10, DAPI mask used to reveal
nuclear content and the localisation of nuclear β-catenin after masking, showing
increased nuclear enrichment of β-catenin in fezf2 mis-expressed embryos. Both
grayscale and pseudo-colour images have been used to show the level of β-catenin
presence in the nucleus. (a and a’) control; (b and b’) fezf2 injected embryos. Arrowhead: blastopore lip. (O-P) Axis duplication assay. (O) Schematic
representation of ventral blastomere injection. 250 pg of mRNA, together with
nuclear β-gal mRNA as tracer, was injected into one of the ventral blastomere
(arrowhead) at the 4-cell stage. Embryos were fixed at approximately stage 38 and
stained with Red-gal. D: dorsal side. (P) Results of axis duplication assay. (a) Control
embryo, nuclear β-gal mRNA injection only. (b) 250 pg fezf2 mRNA. (c) 250 pg β-
catenin mRNA. (Q) Dual-luciferase assay on control FOPFlash plasmid containing
mutated TCF-binding sites. pTK-renilla was used as endogenous control. (R) qPCR
analysis on wnt1, wnt3a, and wnt8b in nuclear β-gal (control) or fezf2-injected
neuralised animal cap explants aged to stage 15. n=3 replicates. * P<0.05. In all cases,
error bars represent ±s. e. m. (K) Schematic representation of animal cap explant
assay. mRNA at different combinations were injected at the 1-2 cell stage and allowed
to develop until the mid-blastula stage (stage 8). Animal cap explants were excised at
stage 8 and allowed to develop to stage 20, at which point they were collected for
qPCR analysis. 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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