Roger R. Markwald

Invasion of mesenchyme into three-dimensional collagen gels: A regional and temporal analysis of interaction in embryonic heart tissue☆

In normal heart development the endothelium of the atrioventricular canal, but not the ventricle, produces mesenchymal cells which seed (invade) into the intervening extracellular matrix toward the myocardium at around 64-69 hr of development. We have utilized three-dimensional collagen substrates to examine the initiation of seeding by atrioventricular canal endothelia in vitro and to compare and contrast the responses of the ventricular endothelia. Explants of atrioventricular canals and ventricles from staged embryos were placed on the surfaces of collagen gels prior to the onset of seeding in situ. At varied intervals of incubation, the explant was removed, leaving behind a monolayer on the surface of the gel which consisted of endothelial cells. Subsequently, the endothelial outgrowths were examined for seeded cells. The results confirm the regional endothelial differences seen in vivo. They also show that invasion of the collagen gels is due to an alteration in phenotype mediated by interaction with other components of embryonic heart explant. Lastly, the time course of this tissue interaction in vitro mimics the onset of seeding in vivo.

Migratory behavior of cardiac cushion tissue cells in a collagen-lattice culture system

Abstract A culture system was devised for the study of factors which influence the migration of cardiac cushion tissue cells. Explants of isolated chick atrioventricular canal cushions were placed on hydrated collagen lattices. Cells grew out of the explants from the endocardium and across the surface of the collagen lattices. During further incubation, mesenchyme-type cells seeded from the surface population into the underlying collagen matrix. These cells were morphologically similar to the mesenchymal cushion tissue cells which are derived from the endocardium and which migrate into and through the cardiac jelly matrix in the embryonic heart. The events observed in the culture system mimicked those occurring in the developing chick atrioventricular cushion.

Myocardial specificity for initiating endothelial-mesenchymal cell transition in embryonic chick heart correlates with a particulate distribution of fibronectin

The early chick heart tube consists of myocardium and endothelium separated by a myocardially derived basement membrane (MBM). As development proceeds, the endothelium undergoes a transition into mesenchyme in a regionally specific manner; only the atrioventricular (AV) and outflow tract, but not the ventricular endothelium, is transformed into mesenchyme, the progenitor of heart septa and valves. Recent experiments have shown that an EDTA extract of MBM can initiate AV endothelium to form mesenchyme in an in vitro collagen gel culture system. Two-dimensional gel electrophoresis of AV region EDTA extracts showed potentially three isoelectric forms of fibronectin (Fn), while extracts from ventricle contained only two forms. The purpose of the present study was to further investigate the significance of these regional differences by testing of specific myocardial regions (AV vs ventricle) for their ability to induce endothelium to form mesenchyme in vitro, and to immunohistochemically determine if a regionally specific distribution of Fn exists in the MBM that can be correlated with previous electrophoretic data. Embryonic heart regions cultured on three-dimensional collagen gels showed that AV endothelium could only form mesenchyme if cocultured with AV myocardium. Coculture with ventricular myocardial explants did not initiate differentiation of AV endothelium. In contrast, ventricular endothelial cells did not form mesenchyme when cocultured with AV or ventricle myocardium. Immunohistochemical localization of Fn revealed three distinct morphological patterns of distribution in the AV-MBM, i.e., an intense lamina densa staining, diffuse staining in fibrils, and as particles. The Fn localized in particles (0.1 to 0.5 micron in diameter) appeared as a gradient of decreasing concentration extending from the myocardium toward the endothelium. In contrast, no particulate Fn staining was observed in the ventricular region. EDTA extraction selectively depleted the particulate form of Fn. Previous work has shown that this extract, which contains several lower Mr proteins in addition to Fn, is biologically active in initiating mesenchyme formation from AV endothelium in vitro. These results show that a regionally specific interaction of the myocardium with the endothelium is required to initiate the formation of prevalvular mesenchyme. This interaction may be mediated by a multicomponent complex involving Fn and other proteins which appear as a regionally distinct particulate only in areas of endothelial differentiation.

Expression of smooth muscle alpha-actin in mesenchymal cells during formation of avian endocardial cushion tissue: A role for transforming growth factor β3

During early cardiac morphogenesis, outflow tract (OT) and atrio‐ventricular (AV) endothelial cells differentiate into mesenchymal cells, which have characteristics of smooth muscle‐like myofibroblasts, and which form endocardial cushion tissue, the primordia of valves, and septa in the adult heart. During this embryonic event, transforming growth factor β3 (TGFβ3) is an essential element in the progression of endothelial‐transformation into mesenchyme. TGFβs are known to be a potent inducer for mesodermal differentiation and a promoter for differentiation of endothelial cells into smooth muscle‐like cells. Using a monoclonal antibody against smooth muscle‐specific alpha‐actin (SMA), we examined the immunohistochemical staining of this form of actin in avian endocardial cushion tissue formation. To determine whether TGFβ3 initiates the expression of SMA, the pre‐migratory AV endothelial monolayer was cultured with or without chicken recombinant TGFβ3 and the expression of SMA was examined immunochemically. Migrating mesenchymal cells expressed SMA beneath the cell surface membrane. These cells showed a reduction of endothelial specific marker antigen, QH1. Stationary endothelial cells did not express SMA. The deposition of SMA in the mesenchymal tissue persisted until the end of the fetal period. Pre‐migratory endothelial cells cultured in complete medium (CM199) that contained TGFβ3 expressed SMA, whereas cells cultured in CM199 alone did not. At the onset of the endothelial‐mesenchymal transformation, migrating mesenchymal cells express SMA and the expression of this form of actin is upregulated by TGFβ3. The induction of the expression of SMA by TGFβ3 is one of the initial events in the cytoskeletal reorganization in endothelial cells which separate from one another during the initial phenotypic change associated with the endothelial‐mesenchymal transformation. Dev. Dyn. 209:296–309, 1997.

Induction of an epithelial-mesenchymal transition by an in vivo adheron-like complex☆

The embryonic vertebrate heart consists of two epithelia: the myocardium and endothelium, separated by the myocardial basement membrane (MBM). The myocardium has been shown to induce endothelial transformation into prevalvular mesenchyme in a temporally and site restricted manner. Previously, we hypothesized that the myocardial-endothelial interaction is mediated in vivo by aggregates of 30-nm particles in the MBM which can be removed by EDTA extraction. These MBM extracts contain fibronectin and other lower Mr proteins and can initiate an epithelial-mesenchymal transition in the AV (atrioventricular canal) endothelium of embryonic chick heart in collagen gel culture. These and other data suggested that the 30-nm multicomponent particles are similar, structurally and compositionally, to multimolecular complexes, termed adherons, secreted by L6 muscle cells in culture. The purpose of this study was to (1) test whether the removal of the 30-nm particles from MBM extracts of embryonic chick hearts would remove the in vitro biological activity and (2) determine if the fractionated MBM extracts can cause AV endothelial cells to follow the same differentiation pathway observed in vivo by monitoring immunohistochemically the cell surface expression of N-CAM. Results showed that centrifugation of extract at 100,000g for 1 hr produced a supernatant fraction that was unable to initiate mesenchyme formation from AV endothelium. However, the resuspended pellet fraction did initiate differentiation of endothelium into mesenchyme. Conditioned medium from L6 skeletal muscle cultures could not substitute for the EDTA extract of embryonic heart. Endothelial cells undergoing the transition to form mesenchyme, both in vivo and in vitro, showed a concomitant decrease in N-CAM staining. This suggested that the pellet-induced formation of migrating cells in the collagen gels is not the result a novel in vitro phenomenon.

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.

Structural analyses on the matrical organization of glycosaminoglycans in developing endocardial cushions

Abstract Extracellular glycosaminoglycans (GAG) were examined in embryonic rat valvular primordia (cushion tissue) to determine if there are specific, in situ, intermolecular associations of GAG and if the passage of migrating cushion cells alters matrical organization. Precursor incorporation studies and colloidal iron staining controlled by acidified methylation, pH, and polysaccharidase digestion indicated that both hyaluronate (HA) and chondroitin sulfate (CHS) were secreted into the premigratory matrix with the predominant GAG being HA. Premigratory matrix was revealed by scanning electron microscopy after routine fixation as a microfibrillar stroma; addition of cetylpyridinium chloride (CPCL) to the fixative resulted in the retention of an additional matrical component superimposed upon the microfibrillar stroma. TEM analysis of the CPCL-dependent matrix revealed that it was composed of intertwined 3-nm filaments, electron-dense, amorphous material, and 30-nm granules. Collagen-like microfibrils were associated primarily with the filamentous component of the CPCL-dependent matrix. Ultracytochemical results obtained with dialyzed iron binding regulated by pH and polysaccharidase and protease digestion suggests that the 3-nm filaments contain HA and the granules contain both CHS and protein. Commensurate with cushion cell formation and migration, X-ray dispersive analysis and polyanionic histochemical criteria indicated increased deposition of CHS in the postmigratory matrix (i.e., matrix transversed by cells). Ultrastructurally, the CPCL-dependent components of the postmigratory matrix became progressively restructured within the wedge of migrating cells. In contrast to premigratory matrix, fewer 3-nm filaments were evident, while 30-nm granules heavily studded the collagen-like microfibrils. Physical fixation controls confirmed the variations between pre- and postmigratory matrices. These results suggest that modification in the matrix organization of embryonic heart GAG may be correlated with the migration of cushion tissue mesenchyme.

Structural analysis of cell: Matrix association during the morphogenesis of atrioventricular cushion tissue☆

Abstract Translocation of an endocardially seeded cushion cell progeny across a broad acellular expanse of extracellular matrix (ECM) constitutes a fundamental morphogenetic event in the development of atrioventricular (AV) cushion pads, the primordia of membraneous septa and cardiac valves. Transmission, scanning, and high-voltage electron microscopy together with light microscopic examination of living or fixed tissues were utilized to determine if (1) one component of the ECM more than any other interacted with the motility-like appendages of cushion cells in such a manner as to suggest a physical substratum; (2) any ECM components were organized into polarized “tracks” which could serve to guide cells centrifugally; and (3) cell:ECM associations varied among the cells comprising the migratory wave. Results indicated that two morphologically identifiable matrix components, microfibrils and a continuum of solid pleomorphic strands of heterogeneous composition called cetylpyridinium chloride (CPCL)-dependent matrix, comprised the bulk of the premigratory ECM. Contact of the premigratory matrix by cushion cells at the leading edge (pioneer cells) of the migratory wave coincided with modification in composition of the CPCL matrix and alignment of microfibrils into polarized tracks (an event seemingly dependent on motility appendage formation, since cells lacking processes after cytochalasin B treatment had altered track associations). Trailing cushion cells uniformly populated the ECM, never piled up against the myocardium, had no track associations, formed numerous cell to cell associations, and were coated with a granular remnant of disrupted CPCL-dependent matrix. The foregoing data suggest that active in vivo translocation and subsequent stabilization of cushion cells involve alignment and compositional changes in the premigratory ECM, events linked temporally with the passage of pioneer cells.

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.

Initial expression of type I procollagen in chick cardiac mesenchyme is dependent upon myocardial stimulation

Formation of the atrioventricular (AV) mesenchyme is a critical step in early heart development. Endothelial cells are activated and transformed into a mesenchymal population that invades the cell-free myocardial basement membrane. This process can be duplicated in collagen gel culture, where it has been established that myocardium or its secretory products activate the endothelium. The purpose of the present study was to determine when these activated endothelial and/or mesenchymal cells start producing type I collagen in situ. These results were compared to those obtained from a culture model of mesenchyme formation. The production of type I collagen was monitored using a monoclonal antibody (M38) that recognizes the carboxy-terminal propeptide of human type I procollagen. The initial expression of the latter within activated AV endothelial and mesenchymal cells in ovo was 48 hr following activation. Prior to this time, only the myocardium was reactive with M38. AV explants of early hearts on collagen gels revealed staining of activated endothelial and mesenchymal cells with M38 after 48 hr in coculture with myocardial tissue. Explants that were prevented from activating (myocardium removed) never expressed the M38 antigen. Similarly, AV endothelial monolayers grown in the presence of myocardial conditioned medium activated and expressed type I collagen after 48 hr in culture, whereas those grown in standard medium did not. These results establish the initial expression of type I collagen within activated AV endothelium and mesenchyme. In addition, the data suggest that the expression of type I collagen within the AV mesenchyme may be dependent on extrinsic influences that induce the AV endothelium to transform into mesenchyme.