Role Of Numb Family Proteins In Cardiac Morphogenesis

Background and objective:Vertebrate heart is the first organ formed during embryogenesis.The cardiac morphogenesis is a very precise and complex spatio-temporal process,involving a series of key events,and these events are regulated by a numberous of gene networks,changing or disruption of any process will cause heart failure and congenital heart disease(CHD).Numb family protein(NFPs)is a cell fate determinant and there are two members Numb and Numblike(Numbl).Recent studies have shown that deletion of NFPs leads to defects of outflow tract morphogenesis,suggesting that NFPs play an important role in the development of outflow tract,but the mechanism is still unclear.Meanwhile,we also found that the Notch 1 signaling pathway was heterotopically activated in the cardiomyocytes when Numb and Numbl were deleted,suggesting that the function of NFPs may depend on the Notchl signaling pathway during cardiac development.By applying the Cre-loxp system,we got Numb/Numbl myocardial double knockout and NumblNumbl/Notch1 myocardial triple knockout embryos to investigate whether the functions of NFPs were through Notch1 signaling during cardiac trabeculation.Moreover,we also found Hey2 expression was changed in MDKO by applying RNAscope and immuno-fluorescence,we tested Hey2 expression in differents mutants to find the signaling pathway that regulate Hey2 expression pattern.This study was aimed to reveal the functions of NFPs during cardiac outflow tract remodeling and whether NFPs regulating cardiac trabeculation was Notchl signaling pathway depended,and the spatiotemporal expression and regulation of Hey2 during cardiac morphogenesis.At last,our studies will help people to further understand the congenital heart disease,and provide theoretical basis for the treatment and intervention of CHDs.Methods:1.Establishment of gene conditional knockout embryos:We generate the gene conditional knockout embryos by applying the Cre-loxp system.We use Nkx2.5cre/+,Nfatclcre/+ and Tie2-cre to specifically delete target genes in cardiomyocytes,endocardium and endothelial cells,respectively,2.mRNA deep sequencing and Protein array analysis:By applying mRNA deep sequencing and Protein array analysis control and mutant group heart samples and analyzing the results,the genes with different expression and the corresponding signal pathway were identified.3.Mechanism analysis:(1)Using immunofluorescence staining and RNA in situ hybridization to verily the expression of related proteins and genes.(2)Western Blot and Q-PCR to further verify the relevant protein and mRNA expression levels.(3)FGF2 stimulation and embryos were cultured in vitro and stimulated by FGF2 to confirm the effect of related signal pathway on Hey2 expression and expression pattern.Results:1.The EdU labeling was used to detect the caidiomyocytes proliferation in the outfolw tract.It was found that the proliferation of cardiomyocytes in the outflow tract was spatially different,with the unevenly proliferation rates in the Left and Right side outflow tract.In the absence of NFPs,this proliferation pattern was disrupted.2.In the absence of NFPs,the expression of Pitx2 was reduced and its expression pattern in the outflow tract was changed.3.The absence of NFPs leads to a decrease in the number of cardiac neural crest cells,which may caused by the lower proliferation of cardiac neural crest cells in MDKO.4.Progenitor cells in the second heart field was increased with the absence of NFPs and was disorderly arranged.5.Myocardial Notch1 knockout in MDKO can partially rescue the trabeculae defects caused by NFPs deficiency.6.Myocardial Notch1 knockout in MDKO can partially reduce the hyper proliferation of trabecular cardiomyocytes caused by NFPs deficiency.7.Notch1/?-catenin/Isl1 signaling pathway was upregulated in the absence of NFPs and downregulated in Numb,Numb1,Notch 1 triple knockout heart.8.The expression of Hey2 is spatio-temporal ly expressed and regulated during cardiac morphogenesis.9.Notch,Nrg/ErbB2,4 and YAP signaling pathway regulates Hey2 expression during cardiac development,but does not regulate its expression pattern.10.FGF signaling pathway regulates the expression level and expression pattern of Hey2 during cardiac development.Conclusions:1.NFPs regulate the proliferation of cardiomyocytes in outflow tract by influencing the Nodal-Pitx2 signaling pathway,and regulate the proliferation of cardiac neural crest cells and the activities of the second progenitor cell progenitor cells.2.NFPs regulate the formation of cardiac trabeculae through Notch1 signaling pathway.3.The expression of Hey2 in the process of heart development is spatial-temporal dependent,and the FGF signaling pathway regulates the expression level and expression pattern of Hey2.