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Filamin A (FlnA) is a ubiquitous actin binding protein which anchors various transmembrane proteins to the cell cytoskeleton and provides a scaffold to many cytoplasmic signaling proteins involved in actin cytoskeleton remodeling in response to mechanical stress and cytokines stimulation. Although the vast majority of FlnA binding partners interact with the carboxy-terminal immunoglobulin like (Igl) repeats of FlnA, little is known on the role of the amino-N-terminal repeats. Here, using cardiac mitral valvular dystrophy associated FlnA-G288R and P637Q mutations located in the N-terminal Igl repeat 1 and 4 respectively as a model, we identified a new role of FlnA N-terminal repeats in small Rho-GTPases regulation. Using FlnA-deficient melanoma and HT1080 cell lines as expression systems we showed that FlnA mutations reduce cell spreading and migration capacities. Furthermore, we defined a signaling network in which FlnA mutations alter the balance between RhoA and Rac1 GTPases activities in favor of RhoA and provided evidences for a role of the Rac1 specific GTPase activating protein FilGAP in this process. Together our work ascribed a new role to the N-terminal repeats of FlnA in Small GTPases regulation and supports a conceptual framework for the role of FlnA mutations in cardiac valve diseases centered around signaling molecules regulating cellular actin cytoskeleton in response to mechanical stress., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Myxoid degeneration of the cardiac valves is a common feature in a heterogeneous group of disorders that includes Marfan syndrome and isolated valvular diseases. Mitral valve prolapse is the most common outcome of these and remains one of the most common indications for valvular surgery. While the etiology of the disease is unknown, recent genetic studies have demonstrated that an X-linked form of familial cardiac valvular dystrophy can be attributed to mutations in the Filamin-A gene. Since these inheritable mutations are present from conception, we hypothesize that filamin-A mutations present at the time of valve morphogenesis lead to dysfunction that progresses postnatally to clinically relevant disease. Therefore, by carefully evaluating genetic factors (such as filamin-A) that play a substantial role in MVP, we can elucidate relevant developmental pathways that contribute to its pathogenesis. In order to understand how developmental expression of a mutant protein can lead to valve disease, the spatio-temporal distribution of filamin-A during cardiac morphogenesis must first be characterized. Although previously thought of as a ubiquitously expressed gene, we demonstrate that filamin-A is robustly expressed in non-myocyte cells throughout cardiac morphogenesis including epicardial and endocardial cells, and mesenchymal cells derived by EMT from these two epithelia, as well as mesenchyme of neural crest origin. In postnatal hearts, expression of filamin-A is significantly decreased in the atrioventricular and outflow tract valve leaflets and their suspensory apparatus. Characterization of the temporal and spatial expression pattern of filamin-A during cardiac morphogenesis is a crucial first step in our understanding of how mutations in filamin-A result in clinically relevant valve disease., ((c) 2010 Wiley-Liss, Inc.)

It is generally thought that the early pre-tubular chick heart is formed by fusion of the anterior or cephalic limits of the paired cardiogenic fields. However, this study shows that the heart fields initially fuse at their midpoint to form a transitory "butterfly"-shaped, cardiogenic structure. Fusion then progresses bi-directionally along the longitudinal axis in both cranial and caudal directions. Using in vivo labeling, we demonstrate that cells along the ventral fusion line are highly motile, crossing future primitive segments. We found that mesoderm cells migrated cephalically from the unfused tips of the anterior/cephalic wings into the head mesenchyme in the region that has been called the secondary heart field. Perturbing the anterior/cranial fusion results in formation of a bi-conal heart. A theoretical role of the ventral fusion line acting as a "heart organizer" and its role in cardia bifida is discussed., (2005 Wiley-Liss, Inc.)

Regenerative medicine is an emerging, but still poorly defined, field of biomedicine. The ongoing 'regenerative medicine revolution' is based on a series of new exciting breakthrough discoveries in the field of stem cell biology and developmental biology. The main problem of regenerative medicine is not so much stem cell differentiation, isolation and lineage diversity, although these are very important issues, but rather stem cell mobilisation, recruitment and integration into functional tissues. The key issue in enhancing tissue and organ regeneration is how to mobilise circulating stem and progenitor cells and how to provide an appropriate environment ('niche') for their tissue and organo-specific recruitment, 'homing' and complete functional integration. We need to know more about basic tissue biology, tissue regeneration and the cellular and molecular mechanisms of tissue turnover (both cellular and extracellular components) at different periods of human life and in different diseases. Systematic in silico, in vitro and in vivo research is a foundation for further progress in regenerative medicine. Regenerative medicine is a rapidly advancing field that opens new and exciting opportunities for completely revolutionary therapeutic modalities and technologies. Regenerative medicine is, at its essence, an emergence of applied stem cell and developmental biology.

Well after formation of the primary linear heart tube, the mesenchymal cardiac septa become largely myocardial, and myocardial sleeves are formed along the caval and pulmonary veins. This second wave of myocardium formation can be envisioned to be the result of recruitment of cardiomyocytes by differentiation from flanking mesenchyme and/or by migration from existing myocardium (myocardialization). As a first step to elucidate the underlying mechanism, we studied in chicken heart development the formation of myocardial cells within intra- and extracardiac mesenchymal structures. We show that the second wave of myocardium formation proceeds in a caudal-to-cranial gradient in vivo. At the venous pole, loosely arranged networks of cardiomyocytes are observed in the dorsal mesocardium from H/H19 onward, in the atrioventricular cushion region from H/H26 onward, and in the proximal outflow tract (conus) from H/H29 onward. The process is completed at H/H stage 43. Subsequently, we determined the potential of the different cardiac compartments to form myocardial networks in a 3D in vitro culture assay. This analysis showed that the competency to form myocardial networks in vitro is a characteristic of the myocardium that is flanked by intra- or extracardiac mesenchyme, i.e., the inflow tract, atrioventricular canal, and outflow tract. These cardiac compartments can be induced to form myocardial networks by a temporally released or secreted signal that is similar throughout the entire heart. Atrial and ventricular compartments are not competent and do not produce the inducer. Moreover, cardiac cushion mesenchyme was found to be able to (trans-)differentiate into cardiomyocytes in the in vitro culture assay. The combined observations suggest that a common mechanism and molecular regulatory pathway underlies the recruitment of mesodermal cells into the cardiogenic lineage during this second wave of myocardium formation through the entire heart., ((c) 2001 Elsevier Science.)

Living morphogenetic studies show that each definitive ventricle is constructed from different primitive cardiac segments, and each has its specific anatomical features. These ventricular segments are the atrioventricular junction; the primitive inlet segment, part of the primary heart tube, which initially provides the inlets of each ventricle; the primitive outlet segment, which gives rise to both ventricular outlets; and the apical trabeculated regions of the right and left ventricles which grow from the primary heart tube, respectively. In this review, we describe regional pathology based on the relationship of these primitive ventricular components. We propose that the abnormal morphogenesis of one of these segments gives origin to regional ventricular pathology. For example, abnormal embryogenesis of the atrioventricular canal produces malformations of the atrioventricular junctions, such as double inlet ventricle, absence of one atrioventricular connection, and straddling and overriding atrioventricular valves. Similarly, abnormal morphogenesis of the primitive outlet segment gives rise to malformations of the subarterial region of each ventricle, along with the valves guarding these vessels. The principal anatomical features of these malformations of the ventricular inlets and outlets are described, and their possible morphogenesis is discussed. Due to the fact that the apical trabeculated region of each ventricle arises from a separate primitive segment, each ventricle can be identified according to the pattern of its apical trabeculations. This feature is crucial in the elucidation of complex congenital pathology, such as discordant atrioventricular connections.

As classically described, the precardiac mesoderm of the paired heart-forming fields migrate and fuse anteriomedially in the ventral midline to form the first segment of the straight heart tube. This segment ultimately forms the right trabeculated ventricle. Additional segments are added to the caudal end of the first in a sequential fashion from the posteriolateral heart-forming field mesoderm. In this study we report that the final major heart segment, which forms the cardiac outflow tract, does not follow this pattern of embryonic development. The cardiac outlet, consisting of the conus and truncus, does not derive from the paired heart-forming fields, but originates separately from a previously unrecognized source of mesoderm located anterior to the initial primitive heart tube segment. Fate-mapping results show that cells labeled in the mesoderm surrounding the aortic sac and anterior to the primitive right ventricle are incorporated into both the conus and the truncus. Conversely, if cells are labeled in the existing right ventricle no incorporation into the cardiac outlet is observed. Tissue explants microdissected from this anterior mesoderm region are capable of forming beating cardiac muscle in vitro when cocultured with explants of the primitive right ventricle. These findings establish the presence of another heart-forming field. This anterior heart-forming field (AHF) consists of mesoderm surrounding the aortic sac immediately anterior to the existing heart tube. This new concept of the heart outlet's embryonic origin provides a new basis for explaining a variety of gene-expression patterns and cardiac defects described in both transgenic animals and human congenital heart disease., (Copyright 2001 Academic Press.)

Formation of the atrioventricular canal (AVC) results from complex interactions of components of the extracellular matrix. In response to signaling molecules, endothelial/mesenchymal transformations are crucial to normal development of the AVC. Atrioventricular septal defects (AVSDs) can result from arrest or interruption of normal endocardial cushion development. The presence of AVSDs has been associated with chromosome abnormalities, laterality or left-right axis abnormalities, and a variety of syndromes. An AVSD susceptibility gene has been identified in a large kindred with many affected members. Studies of transcription factors and signaling molecules in heart development over the past decade are paving the way for our understanding of the heterogeneous mechanisms of causation of AVSDs.

The trisomy 16 (Ts16) mouse is generally considered a model for human Down's syndrome (trisomy 21). However, many of the cardiac defects in the Ts16 mouse do not reflect the heart malformations seen in patients suffering from this chromosomal disorder. In this study we describe the conotruncal malformations in mice with trisomy 16. The development of the outflow tract was immunohistochemically studied in serially sectioned hearts from 34 normal and 26 Ts16 mouse embryos ranging from 8.5 to 14.5 embryonic days. Conotruncal malformations observed in the Ts 16 embryos included double outlet right ventricle, persistent truncus arteriosus, Tetralogy of Fallot, and right-sided aortic arch. This spectrum of malformations is remarkably similar to that seen in humans suffering from DiGeorge syndrome (DGS). As perturbation of neural crest development has been proposed in the pathogenesis of DGS we specifically focussed on the fate of neural crest derived cells during outflow tract development of the Ts16 mouse using an antibody that enabled us to trace these cells during development. Severe perturbation of the neural crest-derived cell population was observed in each trisomic specimen. The abnormalities pertained to: 1) the size of the columns of neural crest-derived cells (or prongs); 2) the spatial orientation of these prongs within the mesenchymal tissues of the outflow tract; and 3) the location in which the neural crest cells interact with the myocardium. The latter abnormality appeared to be responsible for ectopic myocardialization found in trisomic embryos. Our observations strongly suggest that abnormal neural crest cell behavior is involved in the pathogenesis of the conotruncal malformations in the Ts16 mouse., (Copyright 2000 Wiley-Liss, Inc.)

The heart defect (hdf) mouse is a recessive lethal that arose from a transgene insertional mutation on chromosome 13. Embryos homozygous for the transgene die in utero by embryonic day 10.5 postcoitus and exhibit specific defects along the anterior-posterior cardiac axis. The future right ventricle and conus/truncus of the single heart tube fail to form and the endocardial cushions in the atrioventricular and conus/truncus regions are absent. Because the hdf mouse mutation provided the opportunity to identify a gene required for endocardial cushion formation and for specification or maintenance of the anterior most segments of the heart, we initiated studies to further characterize the phenotype, clone the insertion site, and identify the gene disrupted. Chromosome mapping studies first identified the gene, Cspg2 (versican), as a candidate hdf gene. In addition, an antibody recognizing a glycosaminoglycan epitope on versican was found to be positive by immunohistochemistry in the extracellular matrix of normal wild-type embryonic hearts, but absent in homozygous hearts. Expression analysis of the Cspg2 gene showed that the 6/8, 6/9, and 7/9 Cspg2 exon boundaries were present in mRNA of normal wild-type embryonic hearts but absent in the homozygous mutant embryos. DNA sequence flanking the transgene was used to isolate from a normal mouse library overlapping genomic DNA segments that span the transgene insertion site. The contiguous genomic DNA segment was found to contain exon 7 of the Cspg2 in a position 3' to the transgene insertion site. These four separate lines of evidence support the hypothesis that Cspg2 is the gene disrupted by the transgene insertion in the hdf mouse line. The findings of this study and our previous studies of the hdf insertional mutant mouse have shown that normal expression of the Cspg2 gene is required for the successful development of the endocardial cushion swellings and the embryonic heart segments that give rise to the right ventricle and conus/truncus in the outlet of the looped heart., (Copyright 1998 Academic Press.)

Transformation of atrioventricular canal endocardium into invasive mesenchyme is a critical antecedent of cardiac septation and valvulogenesis. Previous studies by Potts et al. (Proc. Natl. Acad. Sci. USA 88, 1510-1520, 1991) showed that treatment of atrioventricular canal endocardial and myocardial cocultures with TGFbeta3 antisense oligodeoxynucleotides blocked mesenchyme formation. Based on this observation, we sought to: (i) identify the target tissue of TGFbeta3 antisense oligos in this transformation bioassay, and (ii) more clearly define the mechanism of TGFbeta3 function in atrioventricular canal mesenchyme formation. In situ hybridization and immunohistochemistry showed little or no TGFbeta3 mRNA or protein in the atrioventricular canal myocardium or endocardium prior to mesenchyme formation (stage 14; paraformaldehyde fixation). However, by stage 18 transforming atrioventricular canal endocardial cells and mesenchyme as well as myocardium were positive for both TGFbeta3 mRNA and protein. In culture bioassays, atrioventricular canal endocardial monolayers pretreated with antisense phosphorothioate oligodeoxynucleotides to TGFbeta3 did not transform into invasive mesenchyme in response to cardiocyte conditioned medium: the subsequent addition of exogenous TGFbeta3 protein relieved this inhibition. Control cultures without pretreatment or those receiving missense oligos generated similar numbers of invasive mesenchyme in response to cardiocyte conditioned medium. Direct addition of TGFbeta3 protein to atrioventricular canal endocardial monolayers in the absence of cardiocyte conditioned medium resulted in loss of cell:cell associations and stimulated cellular hypertrophy, but did not engender invasive mesenchyme formation or alter endocardial proliferation after 24 h of culture. Similar results were obtained with TGFbeta2 protein, either alone or in combination with TGFbeta3. The results of this study indicate that: (i) atrioventricular canal endocardium expresses TGFbeta3 in response to a myocardially derived signal other than TGFbeta3, (ii) atrioventricular canal endocardial TGFbeta3 functions in an autocrine fashion to elicit selected characteristics necessary for cushion tissue formation, and (iii) TGFbeta3 alone or in combination with TGFbeta2 is insufficient to transform atrioventricular canal endocardium into invasive mesenchyme in culture., (Copyright 1998 Academic Press.)

Endocardial cushion tissue is formed by an epithelial-mesenchymal transformation of endocardial cells, a process which results from an inductive interaction between the myocardium and endocardium within the atrioventricular (AV) and outflow tract (OT) regions of the heart. We report here that a protein previously found to be required for myocardially induced transformation of endocardial cells in vitro, ES/130, is highly expressed within the AV and OT regions not only by myocardial cells, but also by the endocardium and its mesenchymal progeny. Given these findings and others, we have tested the hypothesis that endocardial cushion tissue secretes factors which autoregulate its transformation to mesenchyme. Endocardial cushion tissue was cultured and its conditioned growth medium was harvested and applied to nontransformed endocardial cells maintained in the absence of the inductive myocardium. This treatment resulted in endocardial cell invasion into three-dimensional collagen gels plus increased expression of proteins associated with endocardial cell transformation in vivo. Whereas endocardial cushion tissue was found to express ES/130 protein in vivo and in vitro, minimal detection of ES/130 in its conditioned growth medium was observed in immunoblots. Attempts to inhibit the mesenchyme-promoting activity of the conditioned medium with ES/130 antisense were unsuccessful. However, strong intracellular ES/130 expression was detected in endocardial cells, and this expression correlated with the ability of endocardial cells to transform. For example, the minority of endocardial cultures that failed to transform in response to conditioned medium treatment also failed to undergo increased expression of ES/130. These observations are interpreted to suggest that (i) endocardial cushion tissue secretes factors that promote its transformation to mesenchyme, and (ii) while endocardial cushion tissue appears to signal through secretion of factors other than or in addition to ES/130, intracellular ES/130 expression nevertheless may be a target endocardial cell response required for endocardial cell transformation.

During the early stages of embryogenesis, the mesoderm gives rise to cells of the cardiovascular system which include cardiac myocytes and vascular endothelial and red blood cells. We have investigated the development of these cell phenotypes using aggregate cultures of avian blastoderm cells, which replicated mesodermal cell diversification. The cell phenotypes expressed by the blastoderm cells were dependent upon the age of the blastoderm cells, with Hamburger-Hamilton stage 3 or 4 cells giving rise to endothelial and red blood cells and stage 5 cells producing endothelial and myocardial cells. To begin to understand the stage dependency of the cellular diversification of these aggregate cultures, we treated the cultures with various signaling factors that have been shown to be present in the early avian embryo. These experiments showed that stem cell factor and TGF alpha altered cell phenotypes by stimulating red blood cell and myocardial differentiation, respectively. The ability of these growth factors to shift the differentiation profile of aggregate cultures demonstrated the plasticity of early embryonic cells. To explore the diversification of individual mesodermal cells, labeled QCE-6 cells were incorporated within these blastoderm aggregate cultures. Previous studies have shown that this quail mesodermal cell line possesses characteristics of early nondifferentiated mesodermal cells and can be induced to express either myocardial or endothelial cell phenotypes (C. A. Eisenberg and D. M. Bader, 1996, Circ. Res. 78, 205-216). In the present study, we show that when these cells were cultured as a component of blastoderm cell aggregates, they differentiated into fully contractile cardiomyocytes or endothelial or red blood cells. Moreover, QCE-6 cell differentiation was in accordance with that displayed by the blastoderm cells. Specifically, QCE-6 cells differentiated into red blood cells when cultured within stage 3 or stage 4, but not stage 5, blastoderm cell aggregates. Accordingly, the differentiation of QCE-6 cells into beating cardiomyocytes only occurred when these cells were incorporated into stage 5 blastoderm cell aggregates. The identical sorting and differentiation patterns that were exhibited by QCE-6 and blastoderm cells suggest that expression of differentiated cell types within the early mesoderm is directed by the surrounding environment without immediate cellular commitment. In addition, these results provide further evidence that QCE-6 cells are representative of a multipotential mesodermal stem cell and that they possess the potential to exhibit fully differentiated cell phenotypes.

Valvuloseptal morphogenesis of the primitive heart tube into a four-chambered organ requires the formation of endocardial cushion tissue. The latter is the outcome of an inductive interaction in which endocardial (endothelial) cells are induced to transform into mesenchyme by paracrine signals secreted by the adjacent myocardium. In this study, we propose that transforming endothelial/mesenchymal cells themselves secrete a factor-TGFbeta-3-that functions in an autocrine mode to promote/sustain mesenchyme formation and possibly in a paracrine manner to amplify the original (myocardial) inductive event. Cushion mesenchyme-conditioned medium, previously demonstrated to be an endogenous source of autocrine, migration-promoting factors, was found in the present study to contain TGFbeta-3, as detected by immunoblot analysis. Immunoneutralization of TGFbeta-3 in preparations of cushion mesenchyme-conditioned medium resulted in a failure of treated target endocardial cells to migrate as mesenchyme, whereas inclusion of a control antibody did not inhibit the migration-promoting activity of the conditioned medium. Similar to treatment with the conditioned medium, direct addition of TGFbeta-3 to target endocardial cells also elicited invasive migration but only in cultures which had been activated in vivo by inductive interaction with the myocardium prior to treatment. Selective inhibition of TGFbeta-3-mediated autocrine signaling in continuous cocultures of endocardium plus myocardium resulted in endocardial cells which did not migrate, even though they had expressed early markers associated with endocardial cell activation (e.g., alpha-smooth muscle actin, ES/130, and TGFbeta-3). Collectively, these results suggest that (i) two signaling pathways, myocardial and endocardial, are required to start and complete epithelial-mesenchymal transformation in cushion-forming regions of the heart and (ii) the endocardial pathway signals through iteration of TGFbeta-3 and is not functionally redundant to the myocardial pathway.

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 beta3 (TGF beta3) is an essential element in the progression of endothelial-transformation into mesenchyme. TGF beta(s) 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 TGF beta3 initiates the expression of SMA, the pre-migratory AV endothelial monolayer was cultured with or without chicken recombinant TGF beta3 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 TGF beta3 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 TGF beta3. The induction of the expression of SMA by TGF beta3 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.

All blood vessels are lined by endothelium and, except for the capillaries, surrounded by one or more layers of smooth muscle cells. The origin of the embryonic vascular smooth muscle cell has until now been described from neural crest and locally differentiating mesenchyme. In this study, we have substantial evidence that quail embryonic endothelial cells are competent in the dorsal aorta of the embryo to transdifferentiate into subendothelial mesenchymal cells expressing smooth muscle actins in vivo. At the onset of smooth muscle cell differentiation, QH1-positive endothelial cells were experimentally labeled with a wheat germ agglutinin-colloidal gold marker (WGA-Au). No labeled subendothelial cells were observed at this time. However, 19 hours after the endothelial cells had endocytosed, the WGA-Au-labeled subendothelial mesenchymal cells were observed in the aortic wall. Similarly, during the same time period, subendothelial cells that coexpressed the QH1 endothelial marker and a mesenchymal marker, alpha-smooth muscle actin, were present. In such cells, QH1 expression was reduced to a cell membrane localization. A similar antigen switch was also observed during endocardial-mesenchymal transformation in vitro. Our results are the first direct in vivo evidence that embryonic endothelial cells may transdifferentiate into candidate vascular smooth muscle cells. These data arouse new interpretations of the origin and differentiation of the cells of the vascular wall in normal and diseased vessels.

Formation of the left/right body axis is a critical early step in embryogenesis. The heart loop is one of the first clearly recognizable morphological asymmetries, and the molecular pathway which dictates this laterality is now beginning to be understood. We report here that the left and right precardiac fields of chick differ in their sensitivity to retinoic acid (RA); while RA applied to the right precardiac field at gastrulation randomizes heart looping, left side treatment induces situs inversus only at high RA concentrations. We identified two extracellular matrix proteins, the heart-specific lectin-associated matrix protein-1 (hLAMP1) and the fibrillin-related protein recognized by the antibody JB3, which are distributed asymmetrically within the precardiac fields at the head process stage. In normal embryos, JB3 expression is enhanced within the right precardiac field, and hLAMP-1 is enriched within the left. RA treatment predictably altered the expression of these proteins in a manner consistent with subsequent heart laterality: RA treatments which randomize heart loop direction also equalized or reversed the left/right JB3 and hLAMP-1 distribution prior to heart tube fusion. The existence of asymmetrically expressed extracellular matrix proteins within precardiac regions suggests that interactions between cardiocytes and their environment may contribute to heart laterality determination and looping.

Background: Human embryology textbooks indicate that the trunks of the pulmonary vein and artery originate from the left atrium and aortic sac, respectively, based on histological analyses of limited human specimens. However, our studies show that the pulmonary venous trunk in the mouse as in other nonhuman vertebrates originates from a vascular "sac" at the venous pole, the sinus venosus., Methods: Mouse embryos of 9-11 days gestation were obtained and staged according to Theiler's criteria and fixed in Carnoy's solution. Samples were embedded in paraffin and serial sections were prepared., Results: Histological analysis showed that at day 9.5 the pulmonary venous rudiment was initially observed along the left margin in the extracardiac mesenchyme that separated the venous pole of the heart from the lung buds. The endothelium of the pulmonary vein was continuous, with a vascular sac we identified as sinus venosus based on its location immediately posterior to the left sinoatrial fold. The sinus venosus became incorporated into the left atrium (days 10-10.5) to form part of the posterior atrial wall. Similarly, the pulmonary vein and associated extracardiac mesenchyme were "drawn" into the atrium. This extracardiac mesenchyme of the venous pole, also called "spina vestibuli" and containing the pulmonary vein at its left margin, formed a wedge-shaped invagination within the atrium that contributed nonmuscular tissue to the primary atrial septum., Conclusions: We propose that the orifice of the pulmonary vein establishes a link with the left side of the atrium as a consequence of a venous sac, the sinus venosus, and its associated mesenchyme (in which the root of the pulmonary vein is embedded) being incorporated into the atrium.

The formation of endocardial endothelium in quail embryos was investigated using in vivo and in vitro systems. 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 gets 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, as in 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.

The majority of congenital heart defects arise from abnormal development of valvuloseptal tissue. The primordia of the valve leaflets and membranous septa of the heart are the cardiac cushions. Remodeling of the cushions is associated with a transitional extracellular matrix that includes sulfated proteoglycans and the microfibrillar proteins fibulin and fibrillin. Cushion formation is restricted to the AV canal and ventricular outflow tract regions of the primary heart tube. The proper placement of the cushions may be the result of the development of the primary heart tube as a segmented organ, as well as the subsequent looping of the heart. Segmentation of the heart tube may be demonstrated by the alternating molecular expression pattern along the longitudinal axis. In support of this hypothesis is the restricted expression of BMP-4 and msx-2 to the AV canal and ventricular outflow tract. The importance of looping for cushion positioning may imply that the iv and inv genes and retinoic acid are important for the proper patterning of the heart. The cells of the cushions evolve from endocardial cells that undergo an epithelial-to-mesenchymal transformation. This developmental event is regulated by the myocardium and is probably due to the production of protein complexes, present within the cardiac jelly of the cushion-forming regions, that consist of fibronectin and the ES proteins. Both the cushion mesenchyme and its endocardial cell antecedents express JB3, an ECM protein. JB3 expression is also featured within the heart-forming fields of the primary mesoderm, from which the endocardial progenitors of the cushion cells originate.(ABSTRACT TRUNCATED AT 250 WORDS)

ES/130 is a novel 130-kDa protein that has been linked previously to the transformation of endocardial endothelium into cushion mesenchyme. In the present study we report the localization of protein and mRNA for ES/130 in stages 7-plus through 20 chick embryos and present functional data related to a potential mechanism for ES/130. The temporal and spatial regulation of ES/130 expression suggests that this epithelial-to-mesenchymal transformation is a result of homogenetic induction. Functional studies indicate that myocardially derived ES/130 elicits expression of this protein by target AV endothelial cells, which is linked to a signal transduction cascade. The localization of ES/130 to other sites of inductive interactions (e.g., limb bud ectoderm, gut, and notochord) implies that this protein may have a more widespread importance to embryogenesis beyond its involvement in cardiac cushion tissue formation.