The investigators plan to study the epithelial mesenchymal transformation that occurs in the AV canal and outflow tract. They have prepared a polyclonal antiserum (ES1) against a particulate form of heart ECM (from day 2 embryonic chick hearts). This antiserum recognizes 2 major (28 and 46 kDa) and 3 minor (93, 109, and 180 kDa) proteins that disappear from the heart ECM shortly after the AV mesenchyme cushions fuse (about embryonic day 5). In addition, ES1 antiserum blocks the formation of mesenchyme in vitro. These results suggest that one or more of these 5 proteins play an active role in this process. This proposal seeks to correlate individual ES1 antigens with particular cellular and molecular characteristics of the AV epithelial-mesenchymal transition. Central to their experimental approach is the use of a lambda expression library prepared from embryonic chick heart. The minute quantities of ES1 antigens in embryonic heart tissue prohibits purification of them individually for the production of monospecific antibodies. However, large quantities of ES1-related fusion proteins can be isolated from this expression system, which will serve as substitute immunogens for production of antibodies to the endogenous proteins. Therefore, in order to test their hypothesis that one or more of the ES1 antigens is responsible for eliciting the epithelial-mesenchymal transition of AV endothelium, the investigators propose the following experimental aims: 1)generate monospecific polyclonal antibodies that recognize individual ES1 proteins, 2) evaluate the ability of these monospecific antibodies to block different steps of AV endothelial differentiation in vitro, 3) determine the immunohistochemical localization of relevant ES1 antigens, 4) search for cDNA sequence homologies of ES1 antigens with other proteins, and 5) examine the temporal characteristics of gene expression for key ES1 antigens using in situ hybridization. The distribution of ES1 proteins at other sites of putative inductive interactions within the embryo, e.g. limb ectoderm-mesoderm, will also be examined to assess their global significance.
| Krug, E L; Rezaee, M; Isokawa, K et al. (1995) Transformation of cardiac endothelium into cushion mesenchyme is dependent on ES/130: temporal, spatial, and functional studies in the early chick embryo. Cell Mol Biol Res 41:263-77 |
| Isokawa, K; Rezaee, M; Wunsch, A et al. (1994) Identification of transferrin as one of multiple EDTA-extractable extracellular proteins involved in early chick heart morphogenesis. J Cell Biochem 54:207-18 |
| Nakajima, Y; Krug, E L; Markwald, R R (1994) Myocardial regulation of transforming growth factor-beta expression by outflow tract endothelium in the early embryonic chick heart. Dev Biol 165:615-26 |
| Rezaee, M; Isokawa, K; Halligan, N et al. (1993) Identification of an extracellular 130-kDa protein involved in early cardiac morphogenesis. J Biol Chem 268:14404-11 |
