Fibronectin mediates mesendodermal cell fate decisions

Abstract

Non-cell-autonomous signals often play crucial roles in cell fate decisions during animal development. Reciprocal signaling between endoderm and mesoderm is vital for embryonic development, yet the key signals and mechanisms remain unclear. Here, we show that endodermal cells efficiently promote the emergence of mesodermal cells in the neighboring population through signals containing an essential short-range component. The endoderm-mesoderm interaction promoted precardiac mesoderm formation in mouse embryonic stem cells and involved endodermal production of fibronectin. In vivo, fibronectin deficiency resulted in a dramatic reduction of mesoderm accompanied by endodermal expansion in zebrafish embryos. This event was mediated by regulation of Wnt signaling in mesodermal cells through activation of integrin-β1. Our findings highlight the importance of the extracellular matrix in mediating short-range signals and reveal a novel function of endoderm, involving fibronectin and its downstream signaling cascades, in promoting the emergence of mesoderm.

Keywords: Endoderm; Fibronectin; Integrin-β1; Mesoderm; Wnt.

Fig. 1.

Endoderm-like (End2) cells promote the emergence of mesoderm in ES cells through a short-range signal. (A-C) Co-aggregation of mouse ES (mES) cells with End2 cells during embryoid body (EB) differentiation resulted in increases in (A) the number of EBs with beating foci, (B) the percentage of cardiac troponin T (cTnT)⁺ cells and (C) cardiac gene expression. Improvement in cardiac induction was not observed in EB differentiation with End2-conditioned medium. (D,E) Percentage of brachyury (Bry)-GFP⁺ mesodermal cells in EBs differentiated with End2 cells or End2-conditioned medium. (F,G) FACS plot (F) and quantification (G) of Bry-GFP⁺ cells at day 3 in EBs differentiated with increasing ratios of End2 cell co-culture. (H) Percentage of Bry-GFP⁺ cells at day 3 in co-culture of mES cells with End2 cells, neural stem cells (NSCs) or other mES cells (E14 ESC). (I,J) FACS plot (I) and quantification (J) of Bry-GFP⁺ cells at day 3 in co-culture with smaller numbers of End2 cells, visceral endoderm (VE) or extra-embryonic ectoderm (ExE) isolated from E6.5 mouse embryos at 10:1 ratio of mES cells:End2/VE/ExE. *P<0.05, n≥5 for all experiments. Error bars indicate s.e.m. of biological replicates.

Fig. 2.

End2-induced mesoderm is biased toward precardiac mesoderm. (A) qPCR analysis of relative Mesp1 expression in Bry-GFP⁺ cells isolated from mES^Bry-GFP cells at day 4 with control or End2 cell co-culture. (B) Relative number of Kdr⁺ Pdgfrα⁺ cells among Bry-GFP⁺ cells isolated from control or End2 co-culture. (C) Relative Sox17 expression in Bry-GFP⁺ cells isolated at day 4 from mES^Bry-GFP cells with control or End2 cell co-culture. (D) Percentage of cTnT⁺ cells at day 8 from Bry-GFP⁺ or Bry-GFP⁻ cells isolated from control or End2 co-culture conditions. (E,F) Percentage of GFP⁺ cells (E) or cTnT⁺ cells (F) derived from mES^Nkx2.5-GFP cells differentiated into pre-cardiac mesoderm that was then co-cultured with End2 or control cells as illustrated. *P<0.05, n≥5. Error bars indicate s.e.m. of biological replicates.

Fig. 3.

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, n≥5. Error bars indicate s.e. of biological replicates. KD, knockdown.

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, n≥5. Error bars indicate s.e.m. of biological replicates.