Identification of an extracellular 130-kDa protein involved in early cardiac morphogenesis

Previous studies indicate that the transformation of cardiac endothelium into mesenchyme is dependent upon a developmentally regulated signal expressed by its associated myocardium. This process can be mimicked in culture by substituting a non-cytolytic EDTA extract of embryonic heart tissue for the presence of myocardium. Polyclonal antibodies (ES1) generated against the EDTA-extractable proteins both localized to the cardiac extracellular matrix preceding the transformation of endothelium and blocked this process in culture. Based on these observations, we hypothesized that ES1 antigens participate in the formation of cardiac mesenchyme. The present study was undertaken to prepare cDNA and antibody probes for individual ES1 antigens to better characterize their involvement in this important morphogenetic event. An expression library was constructed in Uni-ZAP using poly(A+) RNA from embryonic cardiocyte cultures that had been shown previously to secrete proteins that engender the formation of cardiac mesenchyme. Screening of this expression library with ES1 antibodies resulted in several clones, one of which ("ES1-2.1a") is described in this report. ES1-2.1a has a 2.6-kilobase pair insert, the sequence of which exhibits no apparent homology to those in data banks. A fragment (852 base pairs) from the 5' region of ES1-2.1a cDNA was subcloned into the expression vector pGEX-2T, and a 20-kDa fragment of the resulting protein used to prepare affinity-purified antibodies. Immunoblotting detected a 130-kDa protein ("ES/130") in two preparations that elicit mesenchyme formation, i.e. EDTA extracts of embryonic hearts and conditioned medium of cardiocyte cultures. Functional studies showed that antibodies to ES/130 inhibited the epithelial-mesenchymal transformation of cardiac endothelium in culture. Immunohistochemistry of cardiocyte cultures localized ES/130 protein to the vacuolar system and secretory granules. By polymerase chain reaction analysis, the message for ES/130 was detected in the developing heart just prior to and during mesenchyme formation. These results are consistent with ES/130 being involved at a critical step in the initiation of the epithelial-mesenchymal transformation of cardiac endothelium.