PTEN Function On Embryonic Early Heart Development

Objective:Endogenous PTEN express in primitive streak during chick embryo gastrulation and mesoderm derived structure later on including heart tubes. We investigated the possibility that PTEN is involved in heart formation on the period of early embryo development.Method:Incubating whole chicken embryo with modified filter paper culture method (EC culture) in vitro at 37℃incubator humidly until embryos developed to stage HH4, Isolating the 25%-40% anterior primitive streak tissue of cardiogenic area selectively in aseptic condition, then move into tissue culture in 10%FCS DMEM-F12 culture medium. About 6～8h of cardiogenic area tiusse begin to grow, after 12 h to record the observation generally,50% of the culturing tissue started to beat spontaneously after incubating 2-5 days at 37℃incubator and eventually they formed one sheet of heart muscle tissue. To prove the tissue to be myocardial tissue, in situ hybridization, immunohistochemistry, Dil tracing and electronic microscopy was employed.Whole un-incubated chicken embryos are cultured using the modified filter paper method at 37℃incubator until the chick embryos to reach HH4 stage, and then transfected PTENs to cardiogenesis area around anterior primitive streak including cardiac stem cells, in which half of transfected embryos were fostered at 37℃for about 30hs continuously until the heart tube formation to study the role of PTEN in the process of cell adhesion and migration; CSC from the other part PTENs-transfected emrbyos were isolated and cultured at 37℃CO2 incubator in vitro until differentiating into myocardial tissue with beating pulse in order to study the role of PTEN in the process of cardiac cell proliferation, differentiation and apoptosis.Result:To prove the tissue to be myocardial tissue successfully, in situ hybridization, immunohistochemistry, DiI tracing and electronic microscopy was employed.The result of whole chick embryos immunohistochemistry (N-cadherin) showed that chick heart tubes transfected by PTEN-WT and PTEN-G129E appeared in reverse cyclization, polycystic malformation and so on while the heart tubes from GFP group and PTEN-C124S group developed roughly normally; After transfection of PTEN, the beating frequency of myocardial tissue cultured in vitro decline faster than GFP group and the beating time was not capable of maintaining as log as control group. There is significantly difference (P<0.05) between PTEN-WT group and the GFP group, while there is no significant difference (P>0.05) among PTEN-WT group, PTEN-C124S group and PTEN-G129E.Conclusion:The tissue which isolated from the 25%-40% anterior primitive streak tissue of cardiogenic area selectively in aseptic condition can differentiate into functional myocardial tissueOur data suggest that PTEN might increase the cell adhesion between the early embryo CSC and cell matrix and interfere with cell migration while promote the differentiation CSC and accelerate the aging of myocardium.