Edward L. Krug

Protein extracts from early embryonic hearts initiate cardiac endothelial cytodifferentiation

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.

AN ANTISERUM (ES1) AGAINST A PARTICULATE FORM OF EXTRACELLULAR MATRIX BLOCKS THE TRANSITION OF CARDIAC ENDOTHELIUM INTO MESENCHYME IN CULTURE

The epithelial-mesenchymal transition of cardiac endothelium is a critical developmental event in the formation of valvular and septal anlagen. We have demonstrated previously that this event can be mimicked in culture by treating atrioventricular canal (AV) endothelium with EDTA-soluble proteins extracted from embryonic heart tissue. This activity was fractionated by ultracentrifugation of the EDTA extract, indicating that the critical proteins existed as a multicomponent complex. Based on these results we propose that: (1) the in vitro particulates in EDTA extracts correspond to an observed particulate form of extracellular matrix within the myocardial basement membrane (MBM) of mesenchyme-forming regions and (2) one or more of the proteins in the MBM particulates function to elicit the epithelial-mesenchymal transition. To test these hypotheses we utilized an antiserum, termed ES1, prepared against EDTA-extractable particulates from embryonic chick hearts. Both ES1 and an anti-fibronectin monoclonal antibody (M3H) co-localized in situ to particles within the MBM; however, no ES1 reactivity towards fibronectin could be detected by ELISA or immunoblot analysis. The ES1-positive MBM particulates were removed by extraction with EDTA, but not with PBS, indicating a divalent cation-mediated association of the constituent proteins. ES1 antibodies recognized two major (28 and 46 kDa) and three minor (93, 109, and 180 kDa) proteins on immunoblots of EDTA-extractable proteins. When tested in culture, ES1 antiserum inhibited the formation of mesenchyme from AV endothelium in a dose-dependent manner, while M3H did not. These results are consistent with an active role for one or more of the ES1 antigens in initiating the formation of AV mesenchyme. The localization of ES1 antigens to the extracellular matrix at other dynamic interfaces, e.g., ectoderm/neural tube and limb bud ectoderm/mesoderm, point to a potentially general importance of ES1 antigens in mediating similar developmental interactions.

Inductive interactions in heart development. Role of cardiac adherons in cushion tissue formation.

Heart development proceeds from a number of causally antecedent events. One of these is the formation of intracardiac mesenchyme or “ endocardial cushion tissue .” The significance of cushion mesenchyme is not so much its size or mass, which is relatively small compared to other cardiac tissues, but its strategic location. Cushions form protrusions in both inlet and outlet limbs of the U-shaped, primary heart tube that function as efficient, primitive valves. The eventual fusion of opposing cushion pads across the lumen of the arterioventricular (AV) canal forms a wedge of mesenchyme (the septum intermedium) that serves to “glue” and guide the union of internal muscular septa.’,* Accordingly, the formation of cushion tissue has important reievance to the etiology of congenital heart disease?