Tcf1 Mediates Wnt/?-catenin Signaling Pathway To Promote Cardiac Differentiation Of Mouse Embryonic Stem Cells

Objective: Wnt/?-catenin signal pathway is a canonical Wnt signal circuitry,which is critical for some biological procedure including differentiation,proliferation,survival,apoptosis,conglutination,hypertrophy and aging.It has been suggested that activation of Wnt/?-catenin signaling pathway could promote mesoderm formation and improve cardiomyogenesis from mouse embryonic stem cells(m ESCs).However,little is known about how Wnt signaling induces cardiac differentiation.Upon stimulation by Wnt ligands,?-catenin is increased in cytoplasm.The stabilized ?-catenin translocates into nucleus,and activates transcription of wnt target genes together with T-cell factor/lymphoid enhancer factor(Tcf/Lef)containing highly homologous mobility group DNA binding domains and ?-catenin interaction domains.Tcf/Lef1 members,including Tcf1,Tcf3,Tcf4 and Lef1,are critical for lineage development.Therefore,our experiment mainly discuss the effect of Tcf/Lef1 transcription factor in Wnt/?-catenin signal circuitry on cardiac differentiaton from m ESCs.Methods: The embryonic bodies(EBs)were formed through suspension culture method,CHIR99021 or Wnt3 a was added into differentiated medium from day 2 to 5,named CHIR99021 group or Wnt3 a group,respectively.In addition,there is a control group in which EBs were automatically differentiated.The expression levels of Brachyury,the mesoderm specific target gene,and Nkx2.5,cardiac-precursor marker,as well as the transcripts of cardiomyocyte markers,?-myosin heavy chain(?-MHC),cardiac troponin T(c Tn T)and connexin-43(Cx43)were analyzed through quantitative RT-PCR.Besides,the cardiac-specific proteins including ?-MHC,c TNT and CX43 were detected by immunofluorescence and Western blot.For overexpression,the m ESCs were transfected with 2?g Piggy Bac inserted with Tcf/Lef1 gene,accompanied by 2 ?g transposon vector,using Lipofectamine LTX and PlusTM Reagent according to the manufacturer's recommendations.The Tcf/Lef1 positive cells were screened by treatment with 2 ?g/ml puromycin for 1 week.For RNA interference,the p LKO.1 based lentiviral vectors embedded with targets were transfected into 293 T cells in combination with VSVG and ps PAX2 plasmids.The m ESCs were incubated in the virus-containing supernatant for 48 hours and then selected by puromycin.The EBs were formed through suspension culture method,and then were plated into 6-well plate after 7 days.We could observe the morphology of beating EBs through an inverted microscope.The expression levels of Brachyury,Nkx2.5,?-MHC,c Tn T and Cx43 were analyzed through quantitative RT-PCR.Besides,the cardiac-specific proteins including ?-MHC were detected by immunofluorescence and Western blot.Results: The m ESCs did differentiate into cardiomyocytes.The expression of Brachyury was substantially augmented by treatment with CHIR99021 and Wnt3 a,showing a peak of expression at day 7.Similarly,CHIR99021 and Wnt3 a dramatically increased the expression levels of Nkx2.5,?-MHC,c Tn T and Cx43 with the time of differentiation,with the expression of target genes in CHIR99021 group and Wnt3 a group were greater than that in control group and CHIR99021 group were higher than Wnt3 a group at day 15(*P<0.05,**P<0.01).Western blot analysis suggested that the expression of ?-MHC in CHIR99021 group and Wnt3 a group were greater than control group,and CHIR99021 group was higher than Wnt3 a group at day 15.The over-expression cells and knockdown cells were constructed successfully through PB vector and p LKO.1 system.We could found that the expression of Brachyury in Tcf1/Tcf4/Lef1-overexpressing reached peak after 7 differentiation days and then decreased as time went on,besides,the expressions of Nkx2.5,?-MHC,c Tn T and Cx43 were augmented with the time prolonged and reached peak at differentiation 15 day through q RT-PCR,with the Tcf1 be the best of all.(*P<0.05,**P<0.01).Furthermore,the expression of ?-MHC in Tcf1,Tcf4 and Lef1-overexpressing EBs was detected by immunofluorescence staining.Western blot analysis suggested that the expression of ?-MHC was much higher in Tcf1,Lef1 and Tcf4-overexpressing EBs than that in control EBs at day 15,with the Tcf1 being best of all.Conversely,knockdown each Tcf/Lef1 gene could impair the CHIR99021 mediated cardiac differentiation of m ESCs,while loss of Tcf1 is the worst of all.Conclusion: Wnt signaling pathway promotes cardiomyogenesis from m ESCs mainly via interacting Tcf1 factor.Our results may be benefit for an improvement in the future generation and application of ESCs in cell therapy for cardiovascular diseases.