Figure 2

nradd acts as an inhibitor of Wnt/β-catenin signaling. (A) Wnt/β-catenin reporter activity is reduced in 7xTcf:mCherry embryos injected with 250 pg nradd mRNA. (n (tailbud): control 26/26 embryos, nradd 27/31 embryos; n (10-somite): control 26/26 embryos, nradd 25/26 embryos, n (22 hpf): control 21/21 embryos, nradd 29/30 embryos). Three independent experiments were conducted. (B) Classification of phenotypes at 24 hpf caused by wnt8 overexpression in hs:Wnt8a transgenic embryos after 1 hour of heat shock during gastrulation. Class 2: no eyes, reduced forebrain, normal midbrain–hindbrain boundary (mhb, arrow), class 3: no forebrain, severely reduced midbrain, no mhb, class 4: abnormal notochord development. (C) Phenotypes in wild-type (wt) or hs:Wnt8 transgenic embryos injected with nradd mRNA (150 pg) or equimolar amounts of control mRNA heat shocked at shield stage during gastrulation and scored at 24 hpf. Nradd rescues Wnt8-induced phenotypes. (n (tailbud): control 53 embryos, nradd 46 embryos, nradd+hs:Wnt8a 56 embryos, hs:Wnt8a 45 embryos). Three independent experiments were conducted. (D–F) Expression levels of direct Wnt-target genes axin2, cdx4 and sp5l determined by qPCR in nradd-overexpressing wt embryos (nradd) relative to those in wt embryos injected with GFP mRNA (control) and in nradd-overexpressing hs:Wnt8 embryos (hs:Wnt8+nradd) relative to those in hs:Wnt8 embryos injected with GFP mRNA (hs:Wnt8). All target genes are reduced by nradd in both wt and hs:Wnt8 embryos. Statistical significance was evaluated using an unpaired t-test. * p < 0.05, ** p < 0.01 and *** p < 0.001. Error bars represent ± standard deviation (SD, n = 3). Three independent experiments were conducted. (G–H) Average and SD of the mean (error bars) values of pBAR luciferase reporter activity monitoring Wnt/β-catenin signaling activity (normalized to renilla luciferase activity) in (G) HEK293T and (H) SH-SY5Y cells where Wnt/β-catenin signaling is activated by Wnt8 or Wnt3a conditioned media (CM) and transfected with Nradd. Nradd significantly inhibited Wnt signaling in both cell lines. Statistical significance was evaluated using an unpaired t-test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Error bars represent SD. Three independent experiments were performed.