IDENTIFICATION OF AN EXTRACELLULAR 130-KDA PROTEIN INVOLVED IN EARLY CARDIAC MORPHOGENESIS

Previous studies indicate that the transformation of cardiac endotheli um into mesenchyme is dependent upon a developmentally regulated signa l 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 (ES 1) generated against the EDTA-extractable proteins both localized to t he cardiac extracellular matrix preceding the transformation of endoth elium and blocked this process in culture. Based on these observations , we hypothesized that ES1 antigens participate in the formation of ca rdiac mesenchyme. The present study was undertaken to prepare cDNA and antibody probes for individual ES1 antigens to better characterize th eir involvement in this important morphogenetic event. An expression l ibrary was constructed in Uni-ZAP using poly(A+) RNA from embryonic ca rdiocyte cultures that had been shown previously to secrete proteins t hat engender the formation of cardiac mesenchyme. Screening of this ex pression library with ES1 antibodies resulted in several clones, one o f 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 homo logy to those in data banks. A fragment (852 base pairs) from the 5' r egion of ES1-2.1a cDNA was subcloned into the expression vector pGEX-2 T, and a 20-kDa fragment of the resulting protein used to prepare affi nity-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 inhibit ed 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 de veloping heart just prior to and during mesenchyme formation. These re sults are consistent with ES/130 being involved at a critical step in the initiation of the epithelial-mesenchymal transformation of cardiac endothelium.