Fibronectin promotes End2-mediated induction of mesoderm and precardiac mesoderm in vitro and in vivo. (A) Volcano plot of an ECM expression array comparing End2 versus control cells. (B) Transverse sections of E6.5 mouse immunostained for Fn1, Col4, Col1 or Sparc. Arrows indicate visceral endoderm and arrowheads indicate epiblast. (C) Bry-GFP⁺ cells induced by co-culturing mES^Bry-GFP cells with End2 cells containing siRNA knockdown of Fn1, Col4a2, Col4a1, Col1a1 or Sparc. (D) ntl expression (arrowheads) examined by in situ hybridization in zebrafish embryos injected with control or fn1/fn1b morpholinos (MO-fn1/1b) at mid-gastrulation (50% epiboly; dorsolateral view, animal pole on top). (E) gsc expression (arrowheads) examined by in situ hybridization in zebrafish embryos injected with control or MO-fn1/1b at late gastrulation (90% epiboly; dorsal view, animal pole on top). (F) GFP expression in transgenic sox17-GFP zebrafish embryos injected with control or MO-fn1/1b at 50% epiboly (5.5 hpf; dorsal view, animal pole to the top) and at the 9-somite stage (12 hpf; dorsal view, anterior to the left). The dashed line indicates embryo border. (G) Relative number of sox17-GFP⁺ cells at 50% epiboly (5.5 hpf) in control or MO-fn1/1b-injected zebrafish embryos. (H) Relative number of sox17-GFP⁺ cells at the 9-somite stage (12 hpf) in control or MO-fn1/1b-injected zebrafish embryos. (I) Sagittal section of control or MO-fn1/1b morphant sox17-GFP zebrafish embryos at 50% epiboly (5.5 hpf) after in situ hybridization for expression of ntl (black); immunofluorescence indicates sox17-GFP⁺ cells in the ntl expression domain after fn1/1b knockdown. (J) Relative level of Sox17 mRNA in mES cells co-cultured with control or Fn1-deficient End2 cells. (K) Percentage GFP⁺ cells induced in mES^Nkx2.5-GFP cells at day 6 by co-culture with control or Fn1-deficient End2 cells. (L) Relative number of Kdr⁺ Pdgfrα⁺ precardiac mesoderm cells induced at day 4 by control or Fn1-deficient End2 cells. (M) cTnT and DAPI (nuclei) staining of day-8 replated Bry-GFP⁺ mesoderm isolated from co-culture of mES^Bry-GFP cells with control End2 or Fn1-deficient End2 cells at day 4. (N) Percentage of cTnT⁺ cells in M quantified by FACS. (O) Dorsal views of gata5 expression in zebrafish embryos at the 10-somite stage, assayed by in situ hybridization after control or fn1/1b knockdown. pcm, pre-cardiac mesoderm; lpm, lateral plate mesoderm. (P) Dorsal views of myl7 expression in zebrafish embryos at the 16-somite stage, assayed by in situ hybridization after control or fn1/1b knockdown. Cardiac progenitors are indicated by arrows. *P<0.05, ne5. Error bars indicate s.e. of biological replicates. KD, knockdown.

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.

Fibronectin promotes End2-mediated induction of mesoderm and precardiac mesoderm in vitro and in vivo. (A) Knockdown efficiency of the indicated siRNAs assayed by qPCR. (B) Percentage of Bry-GFP+ cells in day-4 EBs cultured with End2 cells with control (IgG) or anti-Fn1 antibody. (C) EBs with increasing ratio of End2 cells stained with DAPI and Fn1 antibody. (D) Adherent culture of mESBry-GFP EBs plated with End2 cells and stained for Fn1 (red) and GFP (green). (E) End2 cells 48 hours after transfection with scrambled siRNA or siRNA against Fn1, stained for Fn1 (green) and β-catenin (red). (F) End2 cells 48 hours after transfection with scrambled siRNA or siRNA against Fn1, stained for pan-cadherin (green) and aPKC (red). (G) EBs containing End2 cells transfected with scrambled siRNA or siRNA against Fn1, stained for pan-cadherin (green) and aPKC (red). (H) FACS analysis of cell proliferation as assayed by Ki-67 or phosphohistone H3 (PH3) staining among Bry-GFP+ cells in EBs aggregated with control or fibronectin-deficient End2 cells. (I) FACS analysis of cell death as assayed by propidium iodide (PI)+ and annexin V staining among Bry-GFP+ populations in EBs aggregated with control or fibronectin-deficient End2 cells. (J) Expression of the pluripotent genes Oct4 and Nanog in day-2 EBs with control or Fn1-deficient End2 cells. (K) Scatter plot of ECM mRNA array from EBs co-cultured with control or Fn1 knockdown End2 cells. (L) Whole-mount in situ hybridization of ntl in control or Fn1/Fn3 knockdown (MO-Fn1/3) zebrafish embryos at 90% epiboly (9 hpf). *P<0.05, error bars indicate s.e.m.