Mechanism Of Platelet-derived Growth Factor Receptor Knockout In Neural Crest Cells Leading To Persistent Truncus Arteriosus

BackgroudNeural crest cells(NCCs)are a a group of pluripotent migrating cells originated from the dorsal neural tube.During the embryonic development,NCCs participate in the development of many tissues and organs including heart.The NCC subsets involved in cardiac development are called cardiac neural crest cells(CNCCs),which are involved in many important heart development events,such as pharyngeal arch artery remodeling,outflow tract(OFT)separation,valve formation,and heart innervation establishment.During the development,the abnormal cell behaviors of CNCCs(including migration,proliferation and differentiation)often leads to heart structure defects and congenital heart diseases(CHD),including persistent truncus arteriosus(PTA),complete transposition of great arteries(TGA),tetralogy of Fallot(TOF),double outlet of right ventricle(DORV),ventricular septal defect(VSD),and abnormal aortic arch and semilunar valve.The knockout of platelet-derived growth factor receptor(Pdgfr)in neural crest cells can lead to OFT separation disorder and PTA phenotype,but the regulation mechanism and abnormal changes of NCCs during outflow tract separation are not clear.Further study on the mechanism of Pdgfr knockout in neural crest cells leading to PTA in embryo mouse heart may help the researchers of CHD,genetics and developmental biology to establish more accurate causal relationship between cardiac phenotype and NCCs,and greatly promote the research and cognition of CHD.Methods and Results1.Double knockout of Pdgfrα and Pdgfrβ in NCCs leads to OFT separation disorder.In order to study the role and mechanism of neural crest cells in the separation of outflow tract in embryonic mouse heart,we constructed Pax3Cre/+;Pdgfrαfl/fl;Pdgfrβfl/fl genetically engineered mice to double knockout Pdgfrα and Pdgfrβ in NCCs.By observing the phenotype of the heart at different developmental time points from E11.5 to E17.5,we confirmed that the fetal mice of this strain showed cardiac OFT separation disorder at E12.5,and PTA and VSD malformation phenotype at E17.5.All of them could not be born normally due to severe malformation abortion at P0.Further study on cardiac OFT showed that compared with the control group,the polarity of OFT mesenchymal cells was disordered after Pdgfr knockout,which may affect the migration and fusion of bilateral mesenchymal cell cushions to the center to form septal bridge,but there was no significant difference in proliferation and apoptosis.2.Single cell transcriptome sequencing was used to explore the mechanism of outflow tract disjunction in embryonic heart.In order to further clarify the role of NCCs in the formation of OFT septum and the abnormalities after Pdgfr knockout,the traditional pathophysiological methods are limited to the resolution of describing the cellular events and molecular regulatory mechanisms during embryonic development at the tissue level,We used high-throughput and high-resolution single-cell transcriptome sequencing to analyze cardiac OFT tissue cells.The results suggest that in the process of normal OFT septation,both mesenchymal cells and some cardiomyocytes derived from NCCs can differentiate into vascular smooth muscle cells,which can promote the establishment of OFT central septal bridge and the formation of muscular septal remodeling.However,after Pdgfr knockout,the subpopulations of mesenchymal cells directly involved in the formation of septal bridge were completely absent,and the differentiation of cardiomyocytes into vascular smooth muscle cells was also stopped in a transitional state.The expression of genes related to vascular development and remodeling was significantly down regulated,which suggested that Pdgfr knockout of NCCs could lead to the changes of key cell behavior and regulatory genes in the process of cardiac OFT separation dysfunction.Conclusion:Through the above experiments,we obtained the following conclusions:1)Embryonic mouse model with stable PTA phenotype can be constructed by specific double knockout of Pdgfrα and Pdgfrβ in NCCs;2)The change at the tissue level after Pdgfr knockout is mainly manifested in the polarity disorder of cardiac OFT cells,which affects the fusion of mesenchymal cell cushions to form septal bridge structure;3)The change at the cellular level is mainly manifested in the cell differentiation disorder,including the complete absence of mesenchymal cell subpopulations directly involved in the formation of septal bridge,and the differentiation stagnation of myocardial into vascular smooth muscle cells in the state of transitional cells;4)The change at the cellular level is mainly manifested in the changes of ligand-receptor interactions related to OFT separation.These findings explain the important factors of cardiac OFT separation disorder caused by specific knockout of Pdgfr in NCCs at the tissue,cellular and molecular level,and contribute to promote the understanding of the PTA mechanism.