Biological Characteristics Of Mouse Cardiac Neural Crest Cells And The Associated Genes

Conotruncus arteriosus is the conotruncal position including collected truncus arteriosus and partial ventricle involved. Conotruncal defects (CTD) is a kind of complicated heart disease with cyanosis and hypoxemia, including tetralogy of Fallot (TOF), transposition of the great arteries(TGA), double-outlet right ventricle (DORV), persistent truncus arteriosus (PTA), which accounts for 30% of congenital heart disease. Although the mechanism of human conotruncal defects is complicated, studies have shown that connexin43 and cardiac neural crest cells (CNCCs) closely related to conotruncal development. CNCCs migrate from 3, 4, 6 pharyngeal gland to original heart tube, localize at truncus arteriosus and conotruncal region, differentiate into mesenchymal cells that give rise to the formation of outflow tract septum and blood vessel. Heart malformation, such as PTA, DORV, VSD and aortic arch malformation, were detected when neural crest were taken out before they migrated into 3, 4, 6 pharyngeal gland. Interestingly, the types of malformation were related with the length of neural crest destroyed; PTA was detected if the length exceeds two somites, while DORV would be detected if the length less than two somites. These results indicate that CNCCs are very important to the conotruncal development.Cx43 is one member of connexin gene family, which is proved to be closely related to conotruncal development. Right ventricular outflow tract malformatin and obstruction were found in dysfunction of Cx43 gap junction such as Cx43 KO mice, CMV43 mice and FC mice, which indicates appropriate expression of Cx43 plays a very important role in conotruncal development. In the process of mouse embryo development, Cx43 expressed in CNCCs during migration (as early as at ED9.5d), which also take part in gap junction formation of CNCCs. Lots of studies indicate that Cx43 may affect heart development through regulating the biological characteristics of CNCCs.Hence, in this study we used Cx43KO mouse and microarray technology to explore the role of Cx43 in affecting the induction, generation, delamination, migration and differentiation of cardiac neural crest cells, and therefore further reveal the molecular mechanisms of the conotruncal malformations in Cx43KO mice.Part I Isolation, cultivation and biological characteristics analysis of cardiac neural crest cells of embryonic miceObjectiveTo establish the in-vitro culture methods of cardiac neural crest cells of embryonic mice and identify their biological properties, such as proliferation capacity, migration and differentiation potential.MethodsNeural tubes isolated from the region between midotic placode and third somite of mouse embryo at ED 8.5d were cultured in DMEM/F12 serum-free medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The cell cycle of CNCCs was analyzed with flow cytometry, the proliferation capability was determined by MTT method, AP-2α expression was detected by immunocytochemistry as a biological marker of cardiac neural crest cells. The features of migration, multi-directional differentiation were observed as well.ResultsThe MTT and cell cycle analysis indicated that cells proliferated actively. AP-2α expressed abundantly in the cultured neural crest cells, which was of the ability of migration and was able to differentiate into neurons and astrocytes as well.Conclusions1. The cardiac neural crest cells of mouse embryo can be successfully cultured in vitro.2. These cultured cells have the ability of proliferation, migration and the potential of multi-directional differentiation.Part II Differentially expressed genes in the Cx43 knockout cardiac neural crest cells of miceObjectiveTo investigate changes of gene expression in the cardiac neural crest cells in Cx43 knockout mouse embryo, and to elucidate the genes involving in the pathogenesis of conotruncal malformation in Cx43 knockout mouse.MethodsThe cDNA was retrotranscribed from RNA extracted from cardiac neural crest cells of both Cx43 knockout and Cx43 wildtype mouse embryos on ED8.5d. The biotin-labeled cRNA derived from the transcription of cDNA was fragmented as probes. The probes were hybridized with Affymetrix Mouse Genome 430 2.0 Array. Gene Array Scanner was used to screen the signals of hybridization and the expression of genes was detected. RT-PCR was utilized to confirm the changes of partial genes that were detected by Affymetrix Mouse Genome 430 2.0 Array.Results1. Among the differently expressed genes, there were 288 upregulated and 124 downregulated in CNCCs of Cx43 knockout mice as compared with those of Cx43 wild type mice. Functions of proteins encoded by the altered genes encompassed all functional categories, with largest percentage in genes involved in regulation of transcription, cell motility, cell cycle and metabolism.2. The data of RT-PCR indicate that cDNA Array results accurately reflect changes in gene expression patterns in cardiac neural crests of Cx43KO mouse as compared with those of WT mouse.ConclusionsBy cDNA array technology, we compare changes in gene expression between CNCCs of Cx43 KO mouse and those of WT mouse, in which genes related to regulation of transcription changed most, indicating Cx43 may afffect the function of cardiac neural crest cells through regulation of transcription.