Genetic Fate Mapping Reveals The Potential Of Tbx18~+EPCs Differentiation Into Cardiomyocyte Lineages In The Mouse Heart

CHAPTER ONETHE BREEDING AND IDENTIFYING OF TBX18-CREKNOCK IN MOUSEGenetically engineered mouse are considered as the best animal modelin understanding the functions of genes and establishing animal model ofhuman genetic disease. It is also the most popular and effective researchmethod study on the development process of embryonic heart andmyocardial injury or repair in whole animal phenotype level in functionalgenomics. Genetic Fate Mapping is a key technology in tracing thedifferentiation fate of cardiac progenitor cells, but the breeding andidentifying of the genetically engineered mice strain requires asophisticated research platform. T-box transcription factor Tbx18is one ofthe important transcription factors in T-box lineages, which plays a key rolein regulating the development of mammalian heart. How to trace thedifferentiation fate of the cardiac progenitor cells marked by T-boxtranscription factor Tbx18in developing heart has some technical difficulties. We may solve the problem by establishingTbx18-Cre/Rosa26RYFP/LacZdouble-heterozygous mice model.OBJECTIVE: To optimize the methods of genome extraction andestablish a R^YFP/LacZin heterozygous mice on a large scale in order to supplya key technical guarantee to establishing the double-heterozygous geneticfate mapping model.METHODS: Tbx18-Cre Knock in heterozygous male mouse was matedwith the C57BL/6female mouse to identify and screen the Tbx18-CreKnock in heterozygous offspring by extracting genomic DNA using theethanol method. The quality of genomic DNA was assessed by restrictionendonuclease digestion, PCR amplifications, purity and concentration ofDNA which were electrophoresed through agarose gel andspectrophotometry. According to the heterozygote positive rate to judge thegenetic information whether accord with the Mendelian genetics rule.RESULTS:(1) Gel electrophoresis and spectrophotometry revealed thatthere was no difference between the ethanol method and classical phenol/chloroform extraction method in the quality genomic DNA; with the twoextraction protocols, we were able to identify the transgenic animals byPCR amplification; the genomic DNA obtained by the two extractionprotocols were completely digested.(2) We have identified Tbx18-CreKnock in mice by PCR amplification the genomic DNA obtained by ethanol method, the positive rate was51.6%in the offspring of Tbx18-Cremice, which was consistent with the Mendelian genetics rule. The positiverate of offspring in Rosa26^EYFPand Rosa26^LacZmice also accorded with theMendelian genetics rule approximately.CONCLUSIONS:(1) Extracting DNA through the ethanol method issuperior in quality and has a stable and reliable test result by PCRamplification, the genomic DNA obtained by the ethanol method wascompletely digested. This suggests that the genomic DNA isolated by theethanol method is suitable for Southern blot analysis and so on.Consequently we established a research platform successfully which couldidentify and screen the transgenic mice on a large scale.(2)The identifiedoffspring of Tbx18-Cre, Rosa26^EYFPand Rosa26^LacZheterozygous mousehad not found genetic leakage. The identifying of knock-in mouse providedthe most critical security for the subsequent tests.CHAPTER TWOESTABLISHMENT OF THE MODEL OF THEDIFFERENTIATION FATE MAPPING OF Tbx18⁺CELLS INTHE DEVELOPMENT OF EMBRYONIC HEARTIt's hard to trace the differentiation fate of the cardiac progenitor cellsmarked by T-box transcription factor Tbx18in vivo and in vitro. Weproposed to solve the problem by establishing the Tbx18-Cre/Rosa26RYFP/LacZdouble-heterozygous mice model.OBJECTIVE: The establishment of the genetic fate mapping model wasidentified by the expression of the gene and protein of Tbx18traced cells inTbx18-Cre/Rosa26RYFP/LacZdouble-heterozygous mice.METHODS:(1) We judged the specificity of Cre expression by observingthe abnormal development and lethal effect of the Tbx18Cre/Cre mouse.(2)Tbx18-Cre Knock in heterozygous female mouse was mated with the maleconditional Cre reporter mice Rosa26^EYFPor Rosa26^LacZto identify thedouble-heterozygous offspring of Tbx18-Cre/Rosa26R^EYFP/LacZby PCR.(3)The expression of Tbx18mRNA was detected in embryo by In situhybridization, and the LacZ expression was detected by X-gal staining,EYFP+cells purified by FACS.RESULTS:(1) Tbx18Cre/Cre mice had the characteristics of embryoniclethality and malformations.(2) The expression of Tbx18mRNA wasconsistent with the expression of reporter gene inTbx18-Cre/Rosa26RYFP/LacZdouble-heterozygous mice.(3) Tbx18tracedEYFP+cells could be purified by FACS easily in vitro.CONCLUSIONS:(1) Tbx18Cre mice can be used in establishing themodel of tracing the fate of Tbx18⁺cells for Tbx18Cre/Cre mice had thefunction of knock-in knock-out the transcription of endogenous Tbx18.(2)The specific expression of Cre confirmed that Tbx18-Cre/Rosa26RYFP/LacZdouble-heterozygous mice could trace the differentiation fate of Tbx18⁺Cells accurately and provided a method for solving the problem oftrace the differentiation fate of Tbx18⁺marked cells in vivo.(3) EYFP+cellscould be purified by FACS easily, which provide a good method for tracingthe differentiation fate of Tbx18⁺cardiac progenitor cells in vitro.CHAPTER THREETHE EXPRESSION AND GENETIC FATE MAPPING OFTBX18IN EMBRYONIC HEARTEpicardium of the embryonic heart is a new origin of cardiomyocytes.Though Wt1+Epicardial progenitor cells(EPCs)had the pluripotency ofdifferentiating into cardiomyocyte lineages, it is undefined that whetherTbx18is located in the epicardium or whether there are Tbx18⁺EPCs.It isof great significance to clarify the puzzle of Tbx18⁺EPCs.OBJECTIVE: To clarify whether Tbx18is located in the epicardium ofearly embryonic heart, and then explore whether the Tbx18⁺epicardiumcould differentiate into cardiomyocytes in histological level.METHODS: We detected the location of Tbx18mRNA in early embryonicheart by In situ hybridization; we detected the location of EYFP and LacZby X-gal staining and immunofluorescence in tracing model; we alsodistinguished the nature of Tbx18-Cre/Rosa26R^EYFP/LacZepicardium bydouble-label immunofluorescence and double-label immunohistochemical.RESULTS:(1) Whole-mount RNA in situ hybridization showed that Tbx18mRNA expression was observed within the proepicardium andepicardium of mouse embryos before E11.5; histological analysisdemonstrated that the expression of Tbx18was localized in the epicardiumrather than within the heart before E11.5.(2) whole-mount X-gal stainingon embryos from Tbx18-Cre/Rosa26^LacZmice showed LacZ was expressedwithin the proepicardium and epicardium of mouse embryos before E11.5;histological analysis demonstrated that the expression of LacZ waslocalized in the epicardium rather than within the heart before E11.5.(3)EYFP signals were examined in epicardium from Tbx18-Cre/Rosa26YFPmice before E11.5, and the EYFP protein was not observed in the cardiactissue; Double-labeled immunoflioresence in the proepicardiumdemonstrated these EYFP-expressing tissues were not co-localized withcTNT-positive cells.(4)The LacZ expression present in the atria, ventricle,ventricular septum and cardiac vessel in Tbx18-Cre/Rosa26^LacZembryosafter E11.5. Co-immunostaing revealed that Tbx18expression was notco-stained in cardiomyocytes of LacZ-positive staining, except with theepicardium. LacZ-expressing cells in ventricular septum were formed viaepicardium cells differentiation, but did not from Tbx18-expressing cellswithin heart.CONCLUSIONS:(1) The expression of Tbx18mRNA was detected in theepicardium before E11.5. Moreover, the epicardium of mouse characterizednon-cardiac tissue and Tbx18mRNA were not observed within heart at this stage of embryonic hearts. Thus, Tbx18is one of the markers for theembryonic epicardium.(2) Tbx18-Cre/Rosa26^LacZmice modeldemonstrated that the atria, ventricle, ventricular septum, coronary vesselsand other cardiac tissue can be derived from Tbx18⁺epicardium afterE11.5.CHAPTER FOUREXPERIMENTAL STUDY OF DIFFERENTIATIONPOTENTIAL OF Tbx18⁺EPCs INTO CARDIOMYOCYTELINEAGESCardiac progenitor cells are the cell populations which have thepluripotency of differentiating into cardiomyocyte lineages such ascardiomyocyte, vascular smooth muscle cells and pacemaker cells.Whether Tbx18⁺EPCs have the pluripotency of differentiating intocardiomyocyte lineages is important for identifying it as a cardiacprogenitor cell.OBJECTIVE: To detect whether the Tbx18⁺EPCs have the pluripotencyof differentiating into cardiomyocyte lineages at cellular level.METHODS: We identified the type and character of cells generated fromTbx18⁺EPCs by double-label immunofluorescence inTbx18-Cre/Rosa26R^EYFP/LacZmodel. RESULTS: The cells generated from Tbx18⁺EPCs could expressanticardiac troponin T(cTNT), anti-smooth muscle myosin heavy chain11and HCN4,but couldn't express VE-cadherin.CONCLUSIONS: The cells derived from Tbx18⁺EPCs have thecharacteristic of cardiomyocytes, vascular smooth muscle cells and cardiacpacemaker cells, but not with vascular endothelial cell, therefore,Tbx18⁺EPCs posses the pluropotency that differentiate into cardiomyocytes,vascular smooth muscle cells, cardiac pacemaker cells and othermyocardial lineage cells.CHAPTER FIVETHE INFLUENCE OF TBX18⁺EPCs ON THE HEARTS OFADULT MICEThe adult mouse hearts are the differentiated tissues and there are noevidence could prove its regeneration potential. The features ofcardiomyocyte are different from each other in embryonic, neonatal andadult stage. The research results of embryonic and neonatal mouse hearttissues couldn't represent the adults completely. It need further study toexplore whether the Tbx18⁺epicardium have the potential of differentiatinginto cardiomyocyte lineages in adult mice.OBJECTIVE: To explore the influence of Tbx18⁺epicardium on theformation of different structure in adult mouse hearts, test whether the tbx18⁺epicardium have the potential to differentiate into cardiomyocytelineages in adult heart and judge the amount of adult heart tissue thatgenerated from Tbx18⁺EPCs.METHODS: We tested the expression of LacZ in adult mouse hearts bythe whole-mount and histological X-gal staining analysis; we identified thetissue characteristics of the adult heart Tbx18⁺epicardium by double-labelimmunofluorescence; we isolated the EYFP+cells by FACS inTbx18-Cre/Rosa26^EYFPadult mouse.RESULT: The LacZ/EYFP expressed in the atria, ventricular septum,ventricular wall, coronary vascular and atrioventricular valves of the adultmouse heart; LacZ/EYFP could also co-expressed anticardiac troponin T(cTNT)in tissue; we had isolated three Tbx18-Cre/Rosa26^EYFPadultmouse' heart tissue cells by FACS, the rate of EYFP+cells were31.07%,34.77%and35.98%.CONCLUSIONS: The atria, ventricular septum, ventricular wall, coronaryvascular and atrioventricular valves of the adult heart could be generatedfrom Tbx18⁺EPCs; approximately one third adult heart tissue cells derivedfrom Tbx18⁺EPCs,so Tbx18⁺EPCs involved in the formation of adult hearttissue partially.