Sonic hedgehog signaling pathway regulates the formation and septation of the outflow tract in the embryoic mouse heart

Yao Kaining; Yan Yunan; Zhou Yifan; Shi Liang; Cao Ximei; Zeeshan Rahim; Yang Yanping

doi:10.12307/2026.679

Abstract：BACKGROUND:Second heart field and cardiac neural crest are involved in the formation and septation of the outflow tract.However,the exact regulatory mechanism of the Sonic hedgehog(Shh)signaling pathway in this process have not been clarified. OBJECTIVE:To explore the regulatory role of the components of the Shh signaling pathway in the formation and septation of outflow tract. METHODS:Mouse embryos at embryonic 10-15 days were obtained from pregnant ICR mice,and serial section samples were prepared after paraffin embedding to observe the spatiotemporal expression patterns of insulin gene enhancer binding protein-1(isl-1),sonic hedgehog(Shh),Ptch1,Ptch2,Smoothened,Gli3,and bone morphogenetic protein 2 by immunohistochemical staining and double immunofluorescence staining.The foregut and adjacent mesoderm mesenchyme of mouse embryos at embryonic 10.5 days were dissected and the relationship between the Shh signaling pathway and bone morphogenetic proteins and cells in the second heart field and cardiac neural crest was explored by western blot and co-immunoprecipitation. RESULTS AND CONCLUSION:(1)On embryonic 10-11.5 days,the expression of Shh and Gli3 was seen in the pharyngeal endoderm,whereas the expression of Ptch1,Ptch2,and Smoothened was coupled to the development of respiratory endoderm.The respiratory endoderm formation resulted in an increase in isl-1-positive progenitors in the second heart field and was involved in the development of the outflow tract.Components of the Shh signaling pathway were not expressed in the core mesenchyme of the branchial arch,but appeared in the dorsal wall of the pericardial cavity.(2)During the formation and fusion of the outflow tract endocardial cushion,Ptch1,Ptch2,and Smoothened were expressed in the myocardium,and Gli3 was expressed in the mesenchymal cells of the myocardium and endocardial cushion,as well as in the walls of the aortic and pulmonary trunk.(3)On embryonic 10-11.5 days,isl-1 and bone morphogenetic protein 2 were co-expressed in the foregut endoderm and second heart field.Activating protein 2α-positive cardiac neural crest cells surrounding the aortic arch arteries co-expressed either Gli3 or bone morphogenetic protein 2.Co-immunoprecipitation result showed that activating protein 2α interacted with Gli3 or bone morphogenetic protein 2.These findings indicate that the Shh signaling pathway contributes to second heart field development,and plays variant roles in the formation,migration,and differentiation of the second heart field subpopulations.The Shh signaling pathway has a sustained role in endocardial cushion formation and fusion of the outflow tract.Gli3 and bone morphogenetic protein 2 cooperatively regulate the migration of cardiac neural crest cells.