Cloning and characteristics of the novel H8D8 protein regulating valvuloseptal endocardial cushion tissue formation

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During the early heart development endothelial cells transform into mesenchyme to form valvuloseptal endocardial cushion tissue. In this endothelial-mesenchymal transformation (EMT), endothelial cells are activated by several signals, including BMP and unknown signaling(s) from the adjacent myocardium. To identify the unknown novel signaling molecule(s), which is emitted from the myocardium, we constructed a novel monoclonal antibody H8D8 against antigens that had been obtained from the concentrated myocardial conditioned medium possessing biological activities to induce the EMT. First, we established the immunohistochemical method to identify the H8D8 protein that is expressed in the cultured embryonic myocardium. We made cDNA library form the embryonic chick heart, divided them several groups containing approximately 150 cDNA clones, and transfected the group into COS cell. Resulting COS cells were examined if they were expressing H8D8 protein by means of immunohistochemistry. To dat … More e, we checked more than 10,000 clines but unfortunately we do not get H8D8-positive clone. We also tempted to identify the H8D8 protein by means of proteome analysis. We first separated the concentrated proteins obtained from cultured embryonic cardiomyocytes that are expressing H8D8 protein by 2D-gelelectrophoresis. Cell lysates of the cultured cardiomyocytes were separated two dimensionally, separated proteins were transferred to the membrane and stained by H8D8 antibody. At this time we did not obtained H8D8-positive spot in this 2D-immunoblot analysis. Further experiments are necessary to identify the H8D8 antigen. We also examined the downstream region of the BMP signaling during the EMT and clarified that both Msx1 and intracellular signaling cascade of Rho-ROCK pathway are important in the regulation of EMT. We further examined the developmental process of coronary artery stem that is established in the conotruncal cushion regions and showed several anlagen of the coronary stems develops, subsequently they fuse each other and establish coronary artery stem. Less