Tbx18~+CPCs Differentiate Into HCN4~+Sinus Node Pacemaker Cells

PartⅠThe found of Tbx18genetic lineage tracing and knock outmodelsObjective: It’s quite a difficulty in tracing the differeniation ofTbx18+proepicardial cardiac progenitor cells during the development ofembryon, thus we found two kinds genetic lineage tracing models Tbx18：Cre/Rosa26EYFP/Laczdouble heterozygous, were found by Cre-loxP system,in order to describe the expression of Tbx18+CPCS.However,to investigatewhether the T-box transcription factor Tbx18-deficient effects thedevelopment of siuns node, we found a knock out system Tbx18：Cre/Creto obtain the comparion with morphogenesis of the pacemaker tissues ofthe neogenetic heart.More over, we confirm the available of Tbx18geneticlineage tracing and konck out models through detecting the distinctiveexpressions of nucleinic acid or protein.Methods: In order to generate double transgenic mice Tbx18：Cre/Rosa26EYFP/Lacz, sexual mature male homozygote reporter genesRosa26EYFP/Laczmice were crossed with female Tbx18：Cre/-heterozygousmice.Genomic DNA prepared from toe biopsies was used for genotyping by qualitation PCR and toe biopsies staining with X-gal using as a markerof gene expression to identify the genotyping. Sexual mature male andfemale mice Tbx18：Cre/-heterozygous were crossed with to generateTbx18-deficient embryon. Using gene copy numbers by RT-PCR,wedistinguish the mutant and wild type mice.Results: According to PCR results,the double heterozygous Tbx18：Cre/Rosa26EYFPexpress Cre and reporter gene Rosa26EYFP.while toebiopsies were stained blue colour represent for double heterozygousTbx18：Cre/Rosa26LacZmice. Here we report the Tbx18genetic lineagetracing and knock out models are consistent with Mendel’s law ofinheritance, which absence of genetic omission and mutation of reportergenes.In short, the two kinds genetic lineage models are able to effectivelymark the Tbx18CPCs and all daughter cells, while maintaining espressionthroughout the lifetime of the cells. While in the mutant model,advanced stage embryons or postnatal stage shortly after24h died ofTbx18-deficient,which are consistent with the gene copy numbers.Conclusions: Tbx18genetic lineage tracing and knock out modelsare successfully found.The double heterozygous mice through monitoringthe expression of Cre effectively revealed the fate of Tbx18-lineage-tracedcells in the heart.And, the mutant model can response to amorphologiaclly change of the development sinus node. PartⅡ Tbx18+CPCs differentiate into sinus node pacemakercellsObjective: The mouse Tbx18+CPCs are pluripotency cardiacprogenitor cells.Understanding the origins and roles of cardiac progenitorcells is inportant for elucidating the pathogenesis of congenital andacquired heart diseases.HCN channels are responsible for Ifin heart.Thedominant HCN transcript is HCN4. The signaling pathways that the cardiacprogenitor cells differentiate into sinus node pacemaker cells haveremained elusive.Thus we investigate the phenomenon of Tbx18+CPCsdifferentiate into sinus node pacemaker cells and the role of Tbx18information of development sinus node.Methods: By monitoring the exactly expression of Cre, we found twokinds genetic lineage tracing systems Tbx18：Cre/Rosa26EYFP/Lacztotracing the Tbx18+CPCs and all daughter cells. And we found that theTbx18+CPCs do give rise to HCN4+pacemaker cells byimmunofluorescence and X-gal stainings with a whole heart and tisssuesections, while comparation of morphologiaclly with the wild orheterozygou mice and mutant mice using HE stainings. Our data from the ECG establish a heart rates change between the wild or heterozygou miceand mutant mice.Results:Using genetic lineage analysis,we identified Tbx18+CPCsdifferentiate into pacemaker cells to form the structurally sinus node,mainly in a large head of SAN and less in a tail. In Tbx18-deficent fetuses,the head of SAN almost lost,which largely decreased HCN4+pacemakercells but it still formed a morphologically normal tail piece,the delay ofSAN myocardium and increase of fibrosis. Our data from the ECGestablish a conclusion that the heart rates is slower in mutant mice than inwild or heterozygou mice.Conclusions: we found that Tbx18+CPCs differentiate into sinus nodepacemaker cells,which mainly formed the large head of SAN. Andcompared with wild or heterozygou mice, the rates of heart in mutant micebecame slower.