Cushion Tissue Research Articles

Stereological study of stage 34 chicken hearts with looping disturbances after retinoic acid treatment: Disturbed growth of myocardium and atrioventricular cushion tissue

Stereological study of stage 34 chicken hearts with looping disturbances after retinoic acid treatment: Disturbed growth of myocardium and atrioventricular cushion tissue

Carbonic anhydrase II expression pattern in mouse embryonic and fetal heart.

Carbonic anhydrase II localization was studied in mouse embryonic and fetal hearts for better understanding of the functions of this enzyme during cardiac organogenesis. Immunocytolabelling was performed on serial sections of frozen hearts after one night's fixation in 4% paraformaldehyde. In the earliest stages studied, 10, 11 and 12 ed (ed = embryonic day; vaginal plug = day 1), a sharp decrease of labelled cells was observed in the endocardium form which cushion-tissue mesenchyme is derived. During the same period, differences in the decreasing frequencies of labelled cells were also observed between three different cushion-tissue mesenchyme localizations: immunostained cells were abundant in the atrioventricular cushions, less numerous in the proximal part of the conotruncal ridges and rare in their distal part. From 13 ed their repartition was more regular along the conotruncus. From 13 to 16 ed the signal was also present in a peculiar region of the myocardium: the anterior and left walls of the left ventricle. At the 18 and 20 ed labelling was found only in some endothelial cells of coronary vessels, particularly in the interventricular septum. The pattern of expression of carbonic anhydrase II in activated endothelial cells and endothelial-derived mesenchyme cells of the cardiac cushion tissue, strongly suggests that this isoenzyme can be a useful marker for a subpopulation of endothelial cells and cells derived from this endothelium that morphologically express signs of active cell behavior (e.g., invasion, migration, proliferation).

Extracellular Fibrillar Structure of Latent TGFβ Binding Protein-1: Role in TGFβ-dependent Endothelial-Mesenchymal Transformation during Endocardial Cushion Tissue Formation in Mouse Embryonic Heart

Transforming growth factor-beta (TGF beta) is a dimeric peptide growth factor which regulates cellular differentiation and proliferation during development. Most cells secrete TGF beta as a large latent TGF beta complex containing mature TGF beta, latency associated peptide, and latent TGF beta-binding protein (LTBP)-1. The biological role of LTBP-1 in development remains unclear. Using a polyclonal antiserum specific for LTBP-1 (Ab39) and three-dimensional collagen gel culture assay of embryonic heart, we examined the tissue distribution of LTBP-1 and its functional role during the formation of endocardial cushion tissue in the mouse embryonic heart. Mature TGF beta protein was required at the onset of the endothelial-mesenchymal transformation to initiate endocardial cushion tissue formation. Double antibody staining showed that LTBP-1 colocalized with TGF beta 1 as an extracellular fibrillar structure surrounding the endocardial cushion mesenchymal cells. Immunogold electronmicroscopy showed that LTBP-1 localized to 40-100 nm extracellular fibrillar structure and 5-10-nm microfibrils. The anti-LTBP-1 antiserum (Ab39) inhibited the endothelial-mesenchymal transformation in atrio-ventricular endocardial cells cocultured with associated myocardium on a three-dimensional collagen gel lattice. This inhibitory effect was reversed by administration of mature TGF beta proteins in culture. These results suggest that LTBP-1 exists as an extracellular fibrillar structure and plays a role in the storage of TGF beta as a large latent TGF beta complex.

Polysialylated NCAM expression on endocardial cells of the chick primary atrial septum

Septation of the tubular heart to form the multi-chambered heart involves endocardial cell mesenchymal transformation at discrete sites. These sites include the crests of endocardial cushions at the atrioventricular junction, crests of the spiral ridges within the outflow tract, and the leading edge of the atrial septum. The factors involved in this multi-step inductive process appear to include the neural cell adhesion molecule (NCAM). The down-regulation of NCAM coincident with mesenchymal transformation has been documented at the atrioventricular cushion tissue. In view of the function-regulating properties of polysialylated NCAM (PSA-NCAM), we hypothesized that this form of NCAM would be playing a role during the dramatic changes in cell-cell interactions occurring in the endocardium at the leading edge of the primary atrial septum. Chicken hearts at stages during primary atrial septum development were fixed with paraformaldehyde and either immunofluorescently stained for the light microscope analysis or immunoperoxidase stained for ultrastructural analysis. A monoclonal antibody to an NCAM polypeptide epitope (5E) was used to detect all forms of NCAM, while a monoclonal to the polysialic acid (5A5) was used to detect that subset of NCAM which is highly polysialylated (PSA-NCAM). By light microscope level analysis, an increase in immunostaining for NCAM and the appearance of PSA-NCAM was detected on embryonic chicken endocardial cells at the leading edge of the growing atrial septum. The ultrastructural analysis revealed that there is also a change in the pattern of NCAM and PSA-NCAM from a polarized localization to a more ubiquitous distribution over the endocardial cell surface as these cells send out processes, form multiple layers, and sink or move into the underlying extracellular matrix. PSA-NCAM was also detected along cell appositions of cells within the matrix. Both NCAM and PSA-NCAM levels were reduced on cells deep within the matrix. These findings indicate that during primary atrial septation, PSA-NCAM may be deployed on endocardial epithelial cells in order to down-regulate cell-cell interactions and allow the detachment and migration of some of these cells into the underlying matrix. Anat. Rec. 247:71–84 © 1997 Wiley-Liss, Inc.

Polysialylated NCAM expression on endocardial cells of the chick primary atrial septum.

Septation of the tubular heart to form the multi-chambered heart involves endocardial cell mesenchymal transformation at discrete sites. These sites include the crests of endocardial cushions at the atrioventricular junction, crests of the spiral ridges within the outflow tract, and the leading edge of the atrial septum. The factors involved in this multi-step inductive process appear to include the neural cell adhesion molecule (NCAM). The down-regulation of NCAM coincident with mesenchymal transformation has been documented at the atrioventricular cushion tissue. In view of the function-regulation properties of polysialylated NCAM (PSA-NCAM), we hypothesized that this form of NCAM would be playing a role during the dramatic changes in cell-cell interactions occurring in the endocardium at the leading edge of the primary atrial septum. Chicken hearts at stages during primary atrial septum development were fixed with paraformaldehyde and either immunofluorescently stained for the light microscope analysis or immunoperoxidase stained for ultrastructural analysis. A monoclonal antibody to an NCAM polypeptide epitope (5E) was used to detect all forms of NCAM, while a monoclonal to the polysialic acid (5A5) was used to detect that subset of NCAM which is highly polysialylated (PSA-NCAM). By light microscope level analysis, an increase in immunostaining for NCAM and the appearance of PSA-NCAM was detected on embryonic chicken endocardial cells at the leading edge of the growing atrial septum. The ultrastructural analysis revealed that there is also a change in the pattern of NCAM and PSA-NCAM from a polarized localization to a more ubiquitous distribution over the endocardial cell surface as these cells send out processes, form multiple layers, and sink or move into the underlying extracellular matrix. PSA-NCAM was also detected along cell appositions of cells within the matrix. Both NCAM and PSA-NCAM levels were reduced on cells deep within the matrix. These findings indicate that during primary atrial septation, PSA-NCAM may be deployed on endocardial epithelial cells in order to down-regulate cell-cell interactions and allow the detachment and migration of some of these cells into the underlying matrix.

DEVELOPMENTAL EXPRESSION OF TYPE VI COLLAGEN IN THE ATRIO-VENTRICULAR CANAL ENDOCARDIAL CUSHIONS OF THE EMBRYONIC HEART. • 167

The development of the endocardial cushions in the embryonic heart appears to involve the expression of a number of extracellular matrix (ECM) proteins and their receptors. Defects of atrioventricular canal endocardial cushion formation occur most frequently in children with trisomy 21 (Down's syndrome). Genes encoding two of the three chains for type VI collagen, a component of most ECMs, are located on human chromosome 21 within the region thought responsible for the congenital heart defect phenotype associated with trisomy 21. We have examined the expression patterns of type VI collagen in the embryonic mouse heart. An affinity-purified polyclonal type VI collagen antibody was used for immunohistochemical analysis on cryofixed mouse heart sections (d8.0 p.c. to neonatal). Primary antibody was visualized with a FITC-conjugated secondary antibody viewed under fluorescent microscopy. We have determined that type VI collagen is first expressed in the endocardial cushions of the antrioventricular (AV) canal in close association with the endocardium and forming cushion mesenchymal cells. Although type VI collagen is subsequently expressed in other areas of the heart, it remains concentrated in the cushion tissue destined to become the AV valves and membranous septa. This expression pattern is retained in the AV valve leaflets and septa of later stage embryos and neonatal mice. Localization of the three individual type VI collagen chains by in situ hybridization using radiolabeled chain-specific riboprobes on embryonic mouse heart sections correlates well with the observed type VI collagen protein immuno-localization. These data demonstrate that type VI collagen is expressed in the developing AV valves and septa, supporting the molecular genetic studies indicating that type VI collagen is involved in the heart defect phenotype seen in trisomy 21.(Supported by NIH SCOR in Congenital Heart Disease, HL 42266-06).

Formation and Early Morphogenesis of Endocardial Endothelial Precursor Cells and the Role of Endoderm

The formation of endocardial endothelium in quail embryos was investigated usingin vivoandin vitrosystems. Based on the expression of an quail endothelial marker, QH-1, the initial emergence of endothelial precursor cells in the embryo occurs at stage 7+(two somites) in the posterior parts of the bilateral heart forming regions. Cells that expressed the QH-1 antigen were mesenchymal and positioned between the mesodermal epithelium of the heart region and the endoderm. By confocal microscopy, an asymmetrical distribution of QH-1 positive cells was observed between the two heart regions: specifically between 7+and 8−, more precursor cells were seen in the right region than the left. Endothelial precursor cells did not appear outside of the heart forming regions until stage 8−(three somites). Free, mesenchymal-like endothelial precursor cells intrinsic to the heart regions also expressed two extracellular antigens, JB3, a fibrillin-like protein, and cytotactin, both associated with segments of the primary heart tube where endothelial cells “re-transform” back to a mesenchymal phenotype during cardiac cushion tissue formation. Between stages 8 and 9 (four to seven somites), (1) QH-1 positive cells within the heart forming region established vascular-like connections with QH-1 positive cells located outside of the heart region, as initially shown by Coffin and Poole (1988), (2) after fusion of the heart regions, a plexus of QH-1 positive cells was formed ventral to the foregut, and (3) the definitive endocardial lining of the primary heart tube formed directly from the ventral plexus of endothelial precursor cells. Because the QH-1 positive, endothelial precursor cells of each heart forming region were always in close association with anterior endoderm, we sought to determine if the endoderm mediated the formation of precursor cells committed to a cardiac endothelial lineage as reflected by their expression of QH-1, JB3 antigen, and cytotactin. To test this hypothesis, precardiac mesodermal explants were isolated from stage 5 heart forming regions prior to their expressing of either endocardial or myocardial markers and cultured on the surface of collagen gels in the presence or absence of endoderm. In the absence of endoderm, precardiac mesoderm of each stage 5 explant remained epithelial, formed contractile tissue, but did not exhibit any QH-1 positive cells or mesenchymal cells. Conversely, when cocultured with endoderm or endoderm conditioned medium, in addition to the formation of contractile tissue, the explant formed mesenchymal cells. The latter invaded the gel lattice and, asin vivo,expressed QH-1 antigen, JB3 antigen, and cytotactin. These findings suggest that endoderm induces mesoderm of the heart fields to undergo an epithelial to mesenchyme transformation that results in the segregation of myocardial and endocardial precursor cells.

Fibulin-1 and fibulin-2 expression during organogenesis in the developing mouse embryo.

Fibulin-1 and fibulin-2 are extracellular matrix proteins with unique structural features. We used in situ hybridization and immunofluorescence staining to examine the expression of fibulin-1 and fibulin-2 during mouse embryogenesis. Both fibulins have previously been shown to be deposited at sites where polarized cells convert into mesenchyme, during early stages of development of endocardial cushion tissue and in neural crest cells. By Northern blots we confirm that expression of fibulin-2 is particularly high in the developing and newborn heart. We also show that fibulin-2 mRNA and protein remained highly expressed during organogenesis in tissues derived from neural crest mesenchyme. In addition, a locally restricted expression pattern of fibulin-1 and fibulin-2 mRNA and protein at sites of epithelial-mesenchymal interactions was detected in two tissues, the developing tooth and hair follicles. In other tissues where epithelial-mesenchymal interactions occur, fibulin-1 mRNA and its corresponding protein were detected rather uniformly around mesenchymal cells, and no expression of fibulin-2 was noted. Fibulin-1 protein was located in some embryonic epithelial basement membranes. Fibulin-1 mRNA was also expressed in the epidermal layer of brain and in the mesenchyme of choroid plexus and the meninges which surround the spinal cord. Overall, fibulin-2 expression was much more limited than fibulin-1 expression. A very prominent expression of fibulin-2 was seen during early stages of chondrogenesis in all cartilages analyzed. These studies show that the differential expression of the fibulin family contributes to the formation of molecularly distinct extracellular matrices already during early developmental stages of a large number of tissues.

The extracellular matrix proteins fibulin-1 and fibulin-2 in the early human embryo.

Fibulin-1 and fibulin-2, two recently identified extracellular matrix proteins with a homologous domain structure, are known to bind various extracellular ligands and calcium. In this study, they have been localized at the light microscopical level in human embryos of gestational weeks 4-10, using polyclonal antibodies. Identical localization patterns were observed for the two fibulins in most of the tissues. In the heart, the endocardial cushion tissue and endocardium, but not the myocardium, were stained, as were the basement membrane zones and adventitia of blood vessels. Staining was also observed in the perichondrium and calcifying regions of developing bones. Moreover, reactions occurred with the gut subepithelium and epithelial basement membranes of the skin. Differences in staining patterns, however, were observed in various neural structures. Fibulin-1 was prominent in the matrix of the leptomeningeal anlage, in basement membranes of the neuroepithelium and the perineurium of peripheral nerves. Fibulin-2 was detected primarily within the neuropithelium, spinal ganglia and peripheral nerves. The early embryonic expression of both fibulins indicates specific roles during organ development and, in particular, involvements in the differentiation of heart, skeletal and neuronal structures.

Inhibition of outflow cushion mesenchyme formation in retinoic acid-induced complete transposition of the great arteries

Endocardial cushion tissue formation, the primordia of valves and septa, is a critical event in cardiac morphogenesis. Maternally administrated all-trans retinoic acid is known to induce complete transposition of the great arteries (TGA) in the mouse embryo. To address the mechanisms of TGA, the effect of retinoic acid on cushion tissue formation was examined. Using a three-dimensional collagen gel culture model, we performed various types of endothelial-mesenchymal transformation assays of co-cultured endocardium with myocardium obtained from 9.5-day mouse embryonic hearts. In vivo immunohistochemical detections of extracellular matrices, fibronectin and type I collagen, were also performed. Endothelial-to-mesenchymal transformation at the onset of cushion tissue formation was suppressed in the outflow tract of embryos exposed to retinoic acid in culture. This inhibitory effect of retinoic acid was spatially restricted to the outflow tract and reversed by treatment with embryonic myocardial conditioned medium enriched in extracellular inductive molecules. Mesenchyme formation in the outflow tract was inhibited at a lower concentration of retinoic acid (10(-10) mol/l) than that which inhibited the atrio-ventricular canal (10(-7) mol/l) in culture. The fibronectin and type I collagen depositions in pre-migratory outflow tract cardiac jelly in retinoic acid-treated embryonic heart were reduced compared to those in the control. Exogenously applied retinoic acid inhibits outflow tract cushion mesenchyme formation in the embryonic heart with TGA. It is suggested that retinoic acid inhibits the expression of extracellular matrices and inductive molecules synthesized by myocardium in the outflow tract.

Epithelial-mesenchymal transformations in early avian heart development.

Cardiac morphogenesis proceeds from a sequential series of epithelial-mesenchymal transitions which begins by establishing bipotential heart-forming cells and later their segregation into endocardial and myocardial lineages. Cells within each lineage integrate to form two concentric epithelia which inductively interact to transform cells of the inner epithelium, the endocardium, into mesenchymal or 'cushion' cells. Noncardiogenic epithelia (dorsal mesocardium, epicardium, neural ectoderm and coelomic mesothelium) undergo transition into populations of extracardiac mesenchyme that combine over time with cushion tissue to remodel the simple tubular heart into a four-chambered organ. Model systems are described for studying the mechanisms of cardiac-related transformations including primary cultures of precardiac epithelia and a differentiation-inducible, avian stem cell line called QCE-6. Focus is centered on the molecular mechanism by which endocardial epithelium transforms into cushion mesenchyme. Experimental findings are reviewed and interpreted in the context of a hypothetical model that seeks to answer why only some cells within an epithelium transform and whether the transformation process is regulated by intrinsic or extrinsic mechanisms. The model proposes that epithelial cells competent to transform to mesenchyme express characteristic markers including receptors for extrinsic signals secreted by stimulator cells (e.g. myocardium). Candidate extrinsic signals include multicomponent complexes called adherons. If applied directly to cultured endocardium, myocardial adherons but not those secreted by L6 myoblasts, induce changes in gene expression within target endocardial cells for proteases and cell:cell and cell:matrix adhesion molecules that accompanied transformation to mesenchyme. A main component of myocardial adherons has been identified as ES antigens, one of which, ES/130, has been cloned, found to have a novel sequence and in culture assays shown to be required for endocardium to transform to mesenchyme. The spatiotemporal pattern of ES protein expression within the embryo suggests that common mechanisms may exist for embryonic epithelial-mesenchymal transformations.

The development of the atrioventricular junction in the human heart.

The histogenesis of the separation between atrial and ventricular myocardium at the atrioventricular junction in the developing human heart has been investigated immunohistochemically by using monoclonal antibodies specific for atrioventricular cushion tissue, mesenchymal cells, atrial and ventricular myocardium, and myocardium of the primary ring. It was found that the insulation between the muscle masses of atrium and ventricle is established by the fusion of the tissues of the atrioventricular sulcus (located at the epicardial side of the junctional myocardium) with those of the atrioventricular cushions (located at the endocardial side of the junctional myocardium). This process takes place at the ventricular margin of the myocardium of the atrioventricular canal. The separation of atrial and ventricular myocardium starts at approximately 7 weeks of development in the anteromedial portion of the right atrioventricular junction and is largely completed around the 12th week of development. The only remaining myocardial continuity between atrial and ventricular myocardium is the atrioventricular axis of conduction. Our findings show that the nonmuscular part of the developing leaflets of the atrioventricular valves derives from the atrioventricular cushions and that the tissues of the atrioventricular groove do not contribute to the development of these leaflets.

Expression patterns of laminin receptor splice variants α6Aβ1 and α6Bβ1 suggest different roles in mouse development

The alpha 6 beta 1 integrin is a receptor for laminins and is present from early stages of mouse embryogenesis. In the present study we determined the temporal and spatial expression of the two cytoplasmic splice variants of the alpha 6 integrin subunit, alpha 6A and alpha 6B, in the early- and mid-gestation mouse postimplantation embryo using RT-PCR, in situ hybridization, and immunofluorescence. Our results show that alpha 6B is present in the embryo at all stages studied and is expressed before alpha 6A. alpha 6A expression begins in 8.5 day p.c. embryos and is initially exclusively localized to the developing heart. In 8.5 (and 9.5) day p.c. embryos alpha 6A mRNA and protein are present in a gradient in the myocardium of the heart tube from strongest expression in the sinus venosus and in the common atrial chamber to a weakening expression along the ventricle and bulbus cordis. In 10.5 day p.c. embryos this gradient is less evident and in 12.5 day p.c. embryos alpha 6A mRNA and protein are present in comparable amounts between atria and ventricles. Neither alpha 6A nor alpha 6B is present in endocardial cushion tissue. By day 12.5 p.c. alpha 6A expression is also present in the developing epidermis, dental primordia, lens, gonads, and in a few epithelia such as those of the digestive tract. alpha 6B expression is always much more widespread than alpha 6A expression. For example, only alpha 6B is present in the myotome of the somites of 9.5 day p.c. embryos, in the developing central and peripheral nervous systems, and in the nephrogenic system at all stages studied, except after the differentiation of the gonads when alpha 6A is also present. Furthermore, alpha 6B is the only splice variant present on endothelial cells. We also examined the distribution of the beta 4 integrin subunit to determine whether the alpha 6 beta 4 integrin was present during these stages of development. Beta 4 protein was absent in early postimplantation stages but was present in the epidermis and digestive tract of 12.5 day p.c. embryos. These results show a differential distribution of alpha 6A and alpha 6B during mouse development and thus strongly suggest a different function of these splice variants during embryogenesis. Our results point to a possible role for the alpha 6A beta 1 integrin in the development of the myocardium of the developing heart, but not in the migration of endocardial cushion cells, while alpha 6B beta 1 could be important in the developing nephrogenic and nervous systems.

Extracellular Matrix Protein Fibulin-2 Is Expressed in the Embryonic Endocardial Cushion Tissue and Is a Prominent Component of Valves in Adult Heart

Fibulins-1 and -2 are two recently characterized extracellular matrix proteins of a novel protein family. They are found in many tissues in the adult stage, but several differences in their expression patterns were noted in cultured cells and adult tissues. Fibulin-1 is particularly well expressed in the endocardial cushion tissue (ECT) during embryogenesis and we therefore compared the expression patterns of fibulin-1 and -2 in the developing mouse heart. Antibodies against mouse fibulin-1 and -2 were used in immunofluorescence, and the expression of mRNAs were studied with synthetic oligonucleotides by in situ hybridization. A striking codistribution of fibulin-1 and -2 was seen at all stages of the development of the endocardial cushion tissue. Fibulin-2 was found to be a more specific marker for the embryonic ECT than fibulin-1, which was also transiently expressed in cardiac muscle. Immunofluorescence showed that the fibulins remained expressed in valves of the heart in the adult stage. The results suggest a role of the two fibulins in the development of the ECT and in the maintenance of the tensile strength of the cardiac valves.

Formation of the tricuspid valve in the human heart.

Some of the problems concerning the origin of the inlet component of the definitive right ventricle were resolved in a previous study in which we showed it to be derived exclusively from the embryonic right ventricle. Questions remain, however, concerning the relative contributions of endocardial cushion tissue and myocardium to the definitive valvar apparatus guarding the right atrioventricular orifice and the origin of the valvar leaflets. The formation of the tricuspid valve was studied by scanning electron microscopic and immunohistochemical techniques. Concurrent with the development of the right atrioventricular connection, a myocardial ridge forms at the boundary between the atrioventricular canal and the embryonic right ventricle. It grows to become a myocardial gully that funnels atrial blood beneath the lesser curvature of the initial heart tube toward the middle of the right ventricle. Fenestrations in the floor of the gully create an additional inferior opening in the funnel, transforming its initial anterior rim into the septomarginal trabeculation. The septum formed by the fusion of the endocardial ridges of the outflow tract becomes myocardialized in its inferior portion to form, in part, the outlet septum and, in part, the supraventricular crest. The smooth atrial surface of the tricuspid valvar leaflets develops from endocardial cushion tissue. The leaflets become freely movable, however, only after delamination of the tension apparatus within the myocardium. The inferior and septal leaflets derive from the gully and the ventricular septum, their delamination being a single, continuous process. The antero-superior leaflet forms by delamination from the developing supraventricular crest. The leaflets of the tricuspid valve develop equally from the endocardial cushion tissues and the myocardium. The myocardium contributing to the valve comes from two sources, the tricuspid gully complex and the developing supraventricular crest. These findings facilitate the understanding of several congenital malformations.

Cardiac Endothelial Heterogeneity Defines Valvular Development as Demonstrated by the Diverse Expression of JB3, an Antigen of the Endocardial Cushion Tissue

The endothelium of the embryonic vertebrate heart evokes a regional specificity that remains an unexplained phenomenon in cardiac morphogenesis. A restricted population of endothelial cells lining the atrioventricular (AV) canal and proximal outflow tract (OT) transforms into mesenchyme, the reputed progenitor of the valves and membranous septa. The remainder of the cells lining these and other regions of the heart, in particular the ventricle, stay epithelial. At the present time there is no information regarding the determinants for endothelial cell diversity. To investigate the molecular basis for functionally distinct endothelial cell populations, we undertook a search for cell surface proteins within the endocardial cushions of Day 4 chicken embryos that might be sensitive to subtle differences in endothelial cell composition. We theorized that monoclonal antibodies raised against proteins expressed during early valve morphogenesis could provide markers for endothelial subpopulations, thereby assisting our efforts in defining, and determining the origin of, endothelial heterogeneity. In the present study, an in vitro collagen gel culture assay was employed to identify an antibody, JB3, that distinguishes between AV/OT endothelium and ventricular endothelium. Based on this assay, JB3-positive material was associated only with AV/OT endothelia or the mesenchyme derived from these epithelia. Also, a network of JB3-positive fibrillar material was observed within the collagen gel surrounding the explanted cells. The JB3 antigen showed a conspicuous distribution in pregastrulation-stage chicken embryos with immunolabeling observed in the initial primitive streak at 5 hr incubation (stage 2). Subsequent detection in the definitive primitive streak, Hensen's node, and notochord indicate a consistent relationship to midline structures. JB3 antigen also localized to the regions of presumptive precardiac mesoderm and, at later stages, neural crest, somites, and ventral mesocardium. These data suggest that the JB3 antigen may play a role in establishing cardiac endothelial diversity by defining a subpopulation of cells destined to participate in valve formation. Moreover, JB3 may also influence formation of the primary axis and mesoderm structures that form at the midline. Immunochemical analyses showed that JB3 recognizes a polypeptide that migrates near the molecular weight position of fibrillin (350-390 kDa), the extracellular matrix protein linked to the Marfan syndrome. Based on the molecular mass and similar immunostaining patterns in early embryos, we propose that the JB3 antigen is a fibrillin isotype or a fibrillin-associated protein.

Localization of transcription factor GATA-4 to regions of the mouse embryo involved in cardiac development

To clarify the role of transcription factor GATA-4 in mammalian development, we have determined the pattern of expression of GATA-4 in early postimplantation mouse embryos. Using in situ hybridization and immunohistochemistry, we find that GATA-4 RNA and protein are expressed in cells associated with heart development. Intraembryonic expression of GATA-4 RNA is first apparent in coelomic epithelial cells of the primitive streak embryo (approximately 7.0-7.5 days postcoitum). During formation and bending of the heart tube (approximately 8 days postcoitum), GATA-4 RNA and protein are expressed in endocardium, myocardium, and embryonic structures containing precardiac mesoderm such as the septum transversum and intraembryonic coelomic epithelium. By the onset of cardiac septation (approximately 9 days postcoitum), abundant GATA-4 RNA expression is evident in endocardium, endocardial cushion tissue, and myocardium. Expression of GATA-4 by the myocardium continues through gestation and after birth. The temporal and spacial patterns of GATA-4 expression support a role for this factor in the regulation of cardiac differentiation, analogous to the established role of transcription factor GATA-1 in the regulation of hematopoiesis.

The extracellular matrix glycoproteins BM-90 and tenascin are expressed in the mesenchyme at sites of endothelial-mesenchymal conversion in the embryonic mouse heart

Abstract BM-90 is a novel glycoprotein initially isolated from the extracellular matrix of a mouse tumor. We here studied the expression of BM-90 during embryonic development of the mouse heart and compared its expression pattern with that of tenascin and laminin. Distribution was studied by immunofluorescence using antibodies specifically raised against mouse BM-90, laminin and tenascin. Some expression of BM-90 was seen in myocardial basement membranes at early developmental stages, but expression abruptly decreased from these sites at day 12 of embryogenesis. Laminin B chains were also found in the muscle basement membranes early but did not decrease with advancing development. The most striking observation was the markedly enriched expression of BM-90 in the endocardial cushion tissue (ECT). The ECT is derived from mesenchymal cells converted from endothelium and they will form the cardiac valves and septa. In the ECT, BM-90 showed considerable co-distribution with tenascin, but tenascin expression was more focal and did not mark all areas of the ECT. Northern blot data show that BM-90 and tenascin were produced by the developing heart. With antibodies detecting A, B1 and B2 chains of mouse laminin, no immunoreactivity was seen in the ECT. Our data thus show clear-cut differences in the molecular composition of the ECT and muscle basement membranes in the developing heart. The focal expression of BM-90 in the ECT suggests that BM-90 could be involved in epithelial-mesenchymal transitions.

Urokinase Production by Embryonic Endocardial-Derived Cells: Regulation by Substrate Composition

Early events in cardiovascular morphogenesis are characterized by cell migrations and extensive tissue remodeling. We are interested in the role played by the extracellular serine protease urokinase in these events. Elevated urokinase activity and mRNA levels have been shown to be associated with the onset of ventricular trabeculation and mesenchymal cell migration in the endocardial cushion tissues of the artioventricular canal and the outflow tract of the quail embryo. In this study, urokinase production by isolated endocardial-derived cells was found to be affected by the composition of the matrix to which the cells were exposed. Interaction of cells with a 45-kDa gelatin-binding fragment of fibronectin upregulated the production of urokinase by nearly threefold. This increase in urokinase activity had profound influences on cell motility and spreading.

RNA and protein localisations of TGFβ2 in the early mouse embryo suggest an involvement in cardiac development

We have performed a detailed analysis of the localisations of RNAs for TGF beta 2 and beta 3, and of TGF beta 2 protein in mouse embryos from 6.5 to 9.5 days post coitum, using in situ hybridisation and immunohistochemistry on serial sections, and whole-mount in situ hybridisation to complete embryos. TGF beta 3 RNA was not seen in any of the tissue sections, but very low levels of the RNA were seen by whole-mount in situ hybridisation around the outflow tract of the heart at 8.5 days post coitum. TGF beta 2 RNA is expressed at high levels in all cells with the potential to differentiate into cardiomyocytes. Additionally, the foregut endoderm, juxtaposed to the heart, and the neuroepithelium at the rostral extremity of the foregut, express very high levels of TGF beta 2 RNA, between 8.5 and 9.5 days post coitum. As cardiomyogenesis proceeds, TGF beta 2 RNA levels diminishes within the myocytes, with a concomitant increase in staining for TGF beta 2 protein. TGF beta 2 protein staining of cardiomyocytes persists throughout development and in the adult, in the absence of detectable levels of the corresponding RNA. Superimposed upon this myocardial pattern of expression, there is an upregulation of TGF beta 2 RNA in the myocardium of the outflow tract and atrioventricular canal between 8.5 and 9.5 days post coitum, which returns to low levels by 11.5 days post coitum. The results are discussed in terms of a potential role of TGF beta 2 in controlling cardiomyogenesis and in inductive interactions leading to cardiac cushion tissue formation.