Wntless/GPR177 Is Involved In Gastrulation And Heart Development In Mice

Chapter Ⅰ Wntless/GPR177 is involved in Gastrulation and Heart Development in MiceBackground:Early embryonic development in mice including gastrulation and organogenesis requires the elegantly orchestrated actions of several essential signaling molecules of Wnt,BMP,Nodal and FGF.A panel of Wnt ligands together with their common downstream target β-catenin,play pivotal role in gastrulation and organ development in mice.The crucial role of Wnt signaling pathway in embryogenesis has repetitively been verified.Aberrant regulation of Wnt family members in embryo render to robust developmental defects.Wnt3 loss-of-function appears to cause the earliest embryo developmental deficiency,which manifests the importance of Wnt signaling in embryo development.While the rich knowledge of Wnt-receiving cells was obtained,knowledge of Wnt-producing is still poor.Wntless/GPR177 has been reported to regulate the maturation,sorting and secretion of Wnt ligands and global deletion of Wntless(Wls)impairs body axis formation.Objective:Investigating how Wntless is involved in mouse gastrulation and heart development.Methods:Remove Wls in the epiblast and cardiac mesoderm and progenitors with several conditional Cre,analyze histological change of Wls-deficient embryos via HE staining,detect specific mRNA expression with Whole mount in situ hybridization,test changes of Wnt signaling pathway,epithelial to mesenchymal transition and cell apoptosis with immunofluorescence staining.Results:This study found that deletion of Wls profoundly hindered the epithelial to mesenchymal transition(EMT)process required for mesoderm migration,which was characterized by an increase in E-cadherin and N-cadherin in Wls-deficient epiblast.While β-catenin protein level was greatly reduced and pattern of β-catenin was greatly changed,indicating Wls plays an important role in maintaining the expression level and pattern of β-catenin in embryos.Furthermore,an increased cell apoptosis was observed in the Wlsdefective epiblast.In addition,Wntless is essential for heart development as deletion of Wls either in the cardiac progenitor or mesoderm resulted in severe heart defects and embryonic lethality.Conclusion:1.Ablation of Wls in the epiblast can result in abnormalities of the embryogenesis due to reduced epithelial to mesenchymal transition,and the process of gastrulation cannot be performed.The embryos die at E8.5.2.Conditional knockout of Wls in the cardiac mesoderm results in severe dysplasia in the embryonic heart at E10.5.3.Conditional knockout of Wls in cardiac progenitor cells showing significant ventricular wall thinning,the embryos die at E14.5.Chapter Ⅱ Heterogeneity of Second Heart Field Derived Cells Contributing to the Wall of Aortic Root and Pulmonary TrunkBackground: Cardiac development begins in the original heart tube formed from the first heart field(FHF),afterwards,progenitors from the second heart field(SHF)migrate into the heart tube,urge the heart tube undergo drastic extension and expansion while undergo rightward looping.Studies in transgene animals showing the cells derived from FHF contribute to the left ventricle and a part of atria,while cells from SHF make contribution to the outflow tract(OFT),right ventricle,part of atria and venous sinus.After a set of septation and remodeling,the OFT develop into ascending aorta and pulmonary trunk,the abnormality of OFT often render to dysplasia of ascending aorta and pulmonary trunk and complex congenital heart diseases.Studies in chicken and mouse embryos showing the difference of SHF progenitors between areas,progenitors from different areas differentiate into cardiomyocytes of pulmonary conus and aortic vestibule,respectively.These works suggest the differentiation direction of SHF derived cells were prefigured before entering into the OFT,but the fate decision regarding the distribution and differentiation of SHF derived cells in two great arteries has not yet been elucidated.Objective: Investigating the spatiotemporal contributing pattern of SHF derived cells to aortic root and pulmonary trunk.Characterizing the assignment of the SHF derived cells into the trunk of aortic root and pulmonary trunk during OFT septation.Investigating the heterogeneity between SHF derived cells in aortic root and pulmonary trunk.Laying the foundation for the exploration of the etiology of congenital heart diseases and new treatment methods.Methods: Mef2c-AHF-Cre and Mef2c-Cre-ERT2 mice were crossed with Rosa26-Tomato/GFP(m Tm G)mice to generate Mef2c-AHF-Cre;m Tm G and Mef2c-Cre-ERT2;m Tm G mice embryos.In order to activate inducible Cre recombination activity,Tamoxifen administration was carried out via intraperitoneal injection,embryos were harvested at E10.5,E11.5,E12.5,respectively.Analyze the distribution of GFP+ cells in OFT or in aortic root and pulmonary trunk.Results: This study found that after the septation of outflow tract,the distribution pattern of Mef2c-AHF+ derived cells in aortic root and pulmonary trunk is almost decided.While there’s a great difference of the distribution pattern of Mef2c-AHF+ derived cells between aortic root and pulmonary trunk.Mef2c-AHF+ derived cells only distributed in the outer 1/3 layer of aortic root.By contrast,Mef2c-AHF+ derived cells distributed in almost whole layer of pulmonary trunk wall.In this study,there’s two waves of Mef2c-AHF+ cells preferring to contribute to aortic root and pulmonary trunk,respectively.One wave of the Mef2c-AHF+ cells migrated earlier into the OFT preferring to contribute to the aortic root,while the other wave of the Mef2c-AHF+ cells migrated later,which tend to contribute to the pulmonary trunk.Conclusion: 1.The distribution patterns of Mef2c-AHF+ derived cells in aortic root and pulmonary trunk are different: Mef2c-AHF+ derived cells only contribute to the outer 1/3 layer in aortic root,while pulmonary trunk is almost absolutely contributed by Mef2c-AHF+ cell derivatives. 2.The Mef2c-AHF+ cells are divided into different subpopulations to participate in the development of aortic root and pulmonary trunk: The fate of Mef2c-AHF+ derived cells were prefigured before the septation of OFT.The Mef2c-AHF+ cells contributing to aortic root migrate into outflow tract before E7.5,while the Mef2c-AHF+ cells contributing to pulmonary trunk migrate later into OFT.