Protein extracts from early embryonic hearts initiate cardiac endothelial cytodifferentiation

Abstract

Prior to the formation of multiple chambers, the embryonic heart consists of two epithelial tubes, one within the other. As development proceeds, portions of the inner epithelium, i.e., the endothelium, undergo a morphological transformation into a migrating mesenchymal cell population. Our results show that this transformation is affected by proteins secreted by the outer epithelium, i.e., the myocardium, into the extracellular matrix between these two tissues. This conclusion is based on tissue autoradiographic studies of whole embryo cultures with 3H-amino acids. Continuous labeling conditions generated an apparent gradient of proteins extending away from the myocardium and contacting the endothelium just prior to the formation of mesenchyme, i.e., activation of the transformation sequence. Pulse/chase studies confirmed this directional movement of matrix protein. By performing sequential extractions of preactivation staged embryonic hearts with EDTA and testicular hyaluronidase followed by ammonium sulfate precipitation we obtained an enriched preparation of cardiac extracellular matrix. This fraction was capable of eliciting several of the events characteristic of endothelial activation in vitro. These events included: (i) cell-cell separation, (ii) lateral cell mobility, and (iii) hypertrophy and polarization of intracellular PAS staining (Golgi apparati). The biological activity of the extract was sensitive to heat denaturation: a homogenate of the remaining extracted tissue would not substitute for the matrix extract. Morphologically the extracted hearts appeared intact, however, the extracellular matrix space was significantly diminished. No more than 6% of the total lactic dehydrogenase activity, a cytosolic enzyme, was found in the extract. Preliminary electrophoretic characterization of the extract (metabolically labeled with 14C-amino acids) indicated that it may contain as many as 35 proteins or subunits. The relationship of ECM to endothelial differentiation in cardiac morphogenesis is discussed as a model for other developmental systems.