Fig. 4

Fibronectin augments mesoderm induction through integrin-dependent activation of Wnt/β-catenin signaling. (A) Percentage of Bry-GFP⁺ cells induced in day-4 mouse EBs cultured with control (IgG) or anti-integrin-β1 (Itgb1) antibody. (B) Relative luciferase activity from a β-catenin/TCF-responsive luciferase construct (TOP-flash) in day-3 EBs with IgG or anti-Itgb1 antibody. The FOP-flash luciferase reporter has a mutation in the TCF binding site. (C) Western analysis for active β-catenin in day-3 EBs exposed to Wnt3a or End2 co-culture in the presence or absence of integrin inhibition with anti-Itgb1 or Fn1 knockdown. (D) Co-immunoprecipitation (IP) of mES cell/End2 co-culture lysate with control or anti-Ilk antibody immunoblotted (IB) with anti-Gsk3β antibody. (E) Western analysis for Ser9-phosphorylated Gsk3β in GFP⁺ cells sorted from day-3 mES^CAG-GFP cells co-cultured with control or Fn1-deficient End2 cells. (F) Relative number of Bry-GFP⁺ mesoderm cells induced by control or Fn1-deficient End2 cells, with or without Wnt3a or the Wnt agonist BIO. (G) In situ hybridization for ntl expression in zebrafish embryos deficient in fn1 and fn1b (MO-fn1/fn1b) at mid-gastrulation (dorsolateral view, animal pole to the top). The ntl expression domain was rescued by activation of Wnt signaling via addition of BIO (0.5 μM) to egg water at 2.5 hpf. The number of observed morphant embryos exhibiting displayed phenotype is indicated. *P<0.05, ne5. Error bars indicate s.e.m. of biological replicates.