Bidirectional fusion of the heart-forming fields in the developing chick embryo.

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.

Cardiopulmonary malformations in the inv/inv mouse.

The inv/inv mouse carries an insertional mutation in the inversin gene, (inv, for inversion of embryonic turning). Previously it had been reported that almost 100% of the homozygous offspring (inv/inv) were characterized by situs inversus totalis. In this report we identify the spectrum of cardiopulmonary anatomical abnormalities in inv/inv mice surviving to birth to determine whether the abnormalities seen are of the categories classically associated with human situs abnormalities. Stillborn mice, offspring that died unexpectedly (within 48 hr after birth), and neonates with phenotypic characteristics of situs inversus (right-sided stomachs, growth failure or jaundice) were processed for standard histological examination. Of 173 offspring, 34 (20%) neonates (11 stillborn, 9 unexpected deaths, and 14 mice with situs inversus phenotype) were examined, 27 of which were genotyped to be inv/inv. Interestingly, three inv/inv mice (11%) were found to have situs solitus. Twenty-four had situs inversus with normal, mirror-image cardiac anatomy (dextrocardia with atrioventricular concordance, ventriculoarterial concordance and a right aortic arch). The overall incidence of cardiovascular anomalies observed was 10 out of 27 (37%). The most frequent severe malformation, identified in 3 out of 27 animals, was a complex consisting of pulmonary infundibular stenosis/atresia with absence of pulmonary valve tissue and a ventricular septal defect. The pulmonary phenotype in inv/inv mice was situs inversus with occasional minor lobar abnormalities. We conclude that 1) cardiopulmonary malformations in inv/inv mice are not rare (37%), 2) the cardiopulmonary malformations observed in inv/inv specimens are not of the spectrum typically associated with human heterotaxia. In particular, inv/inv mice have a propensity for defects in the development of the right ventricular outflow tract and the interventricular septum, and 3) approximately one out of ten inv/inv mice is born with situs solitus and shows cardiac anomalies that correspond to those observed in inv/inv specimens with situs inversus. Our data therefore suggest that inversin, the product of the inv locus, may have specific roles in cardiac morphogenesis independent of its role in situs determination.

Down syndrome congenital heart disease: a narrowed region and a candidate gene.

PURPOSE: Down syndrome (DS) is a major cause of congenital heart disease (CHD) and the most frequent known cause of atrioventricular septal defects (AVSDs). Molecular studies of rare individuals with CHD and partial duplications of chromosome 21 established a candidate region that included D21S55 through the telomere. We now report human molecular and cardiac data that narrow the DS-CHD region, excluding two candidate regions, and propose DSCAM (Down syndrome cell adhesion molecule) as a candidate gene. METHODS: A panel of 19 individuals with partial trisomy 21 was evaluated using quantitative Southern blot dosage analysis and fluorescence in situ hybridization (FISH) with subsets of 32 BACs spanning the region defined by D21S16 (21q11.2) through the telomere. These BACs span the molecular markers D21S55, ERG, ETS2, MX1/2, collagen XVIII and collagen VI A1/A2. Fourteen individuals are duplicated for the candidate region, of whom eight (57%) have the characteristic spectrum of DS-CHD. RESULTS: Combining the results from these eight individuals suggests the candidate region for DS-CHD is demarcated by D21S3 (defined by ventricular septal defect), through PFKL (defined by tetralogy of Fallot). CONCLUSIONS: These data suggest that the presence of three copies of gene(s) from the region is sufficient for the production of subsets of DS-CHD. This region does not include genes located near D21S55, previously proposed as a "DS critical region," or the genes encoding collagens VI and XVIII. Of the potential gene candidates in the narrowed DS-CHD region, DSCAM is notable in that it encodes a cell adhesion molecule, spans more than 840 kb of the candidate region, and is expressed in the heart during cardiac development. Given these properties, we propose DSCAM as a candidate for DS-CHD.

The outflow tract of the heart is recruited from a novel heart-forming field.

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.

Dynamic expression of a native chondroitin sulfate epitope reveals microheterogeneity of extracellular matrix organization in the embryonic chick heart.

TC2 is a novel monoclonal antibody produced by in vitro immunization of splenocytes with a peanut agglutinin-positive fraction from extracts of prechondrogenic micromass cultures of chick limb mesenchyme. ELISA results demonstrated TC2 reactivity with a native epitope on a glycosaminoglycan (GAG) enriched in chondroitin-4-sulfate and with multiple intact proteoglycans, but not with other GAGs tested. TC2 immunohistochemical reactivity was abolished by pretreatment of sections with chondroitinase AC or preadsorption with chondroitin-4-sulfate GAG. Strong TC2 localization occurred throughout the developing heart at stage 9. As looping ensued, a graded reactivity was observed from lowest in the atrium to highest in the conotruncus that correlated well with versican localization. The superior atrioventricular cushion stained preferentially with TC2 as compared to the inferior cushion at stages 16-18. At these later stages TC2 patterns did not agree completely with anti-versican reactivity. By stage 23 there was a marked reduction in TC2 localization in the heart, however, strong reactivity remained at certain sites, including the conotruncus and in subcompartments of both atrioventricular cushions. A heterogeneous distribution of other native chondroitin sulfate glycosaminoglycan epitopes recognized by monoclonal antibodies d1C4 and CS-56 was observed as well. The distribution of the TC2 epitope usually did not overlap with d1C4 or CS-56 localization at the stages examined. Overall, the spatiotemporal characteristics of TC2 reactivity in the developing chick heart appear to correlate with subdomains of the endocardial cushions as well as with trabecular and atrial septal formation.

The Cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation.

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.

Identification of an autocrine signaling pathway that amplifies induction of endocardial cushion tissue in the avian heart.

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.

Expression and function of activin beta A during mouse cardiac cushion tissue formation.

The formation of cardiac cushion tissue, which ultimately contributes to formation of the valves and septa, is dependent on the regional activation of cardiac endothelial cells to undergo an epithelial-mesenchymal transition. This endothelial transition was correlated with activin betaA mRNA expression by Northern and in situ hybridization in both a temporal and spatial manner in developing mouse embryos. Activin betaA was the only subunit of the inhibin family detected during the initial phase of endothelial cell transition; activin betaB was detected at later stages, and inhibin alpha was not detectable in the heart. An in vitro assay that has been used to study mesenchymal cell formation in chick was modified for use with mammalian embryos. Conditioned media from embryonic mouse cardiocyte cultures was shown to substitute for the endogenous inductive signal in these assays. The presence of activin betaA was demonstrated by Western blot analysis of the cardiocyte conditioned media (CCM). Modified antisense oligonucleotides to activin betaA inhibited the endothelial-mesenchymal transition in the assay system, which was not affected by control oligonucleotides. Adapting the avian culture system for use with mice enabled the use of tissue from mice with a null allele for activin betaA. CCM produced from embryos homozygous for the mutant betaA allele did not contain activin betaA and was used in in vitro assays. CCM lacking activin betaA produced fewer mesenchymal cells from cardiac endothelial monolayers than CCM with activin betaA. Localized expression of activin betaA in the embryonic heart indicates a possible role in the endothelial-mesenchymal transition. Bioassays in which activin betaA expression is blocked or activin betaA is absent from the media indicate that activin betaA promotes the formation of mesenchymal cells in the endothelial cushions, which are required for normal septation.

A heart segmental defect in the anterior-posterior axis of a transgenic mutant mouse.

A recessive lethal insertional mutation on chromosome 13 has been identified in a transgenic mouse line that displays a segmental form of cardiac defect along the anterior-posterior axis in all homozygous mice identified. The most anterior segment (future conus and right ventricle) of the single heart tube fails to develop normally and the endocardial cushions in both the conus and the atrioventricular regions are missing. Analysis of the beta-galactosidase reporter portion of the transgene during embryonic development shows a segmental expression of activity primarily in the defective outlet of the primitive heart. In addition to expression in the heart tube, hemizygous embryos show transgene expression in the chondrogenic regions of first and second branchial arches, the appendicular skeleton, and the dermal papillae of the vibrissae. The restricted pattern of beta-galactosidase expression in the heart can be disrupted with retinoic acid exposure and extended posteriorly along the anterior-posterior axis in hemizygous mice. Although cushion mesenchyme fail to form in the homozygous mutant, the myocardial and endothelial cells explanted from the mutant atrioventricular, but not the conus, are capable of forming mesenchyme in vitro. Mice trisomic for chromosome 13 have also been shown to display segmental anomalies associated with the anterior primitive outlet segments of the heart. Our data show that this insertional mutation identifies a new gene locus, hdf (heart defect), on mouse chromosome 13 that may be required for mechanisms that initially establish and/or maintain continued development of the anterior limb of the developing heart. The hdf mouse mutation also provides a new model system to evaluate the molecular requirements of normal endocardial cushion formation and the segmental interactions that form the adult heart.

Cellular localization of the class I alcohol dehydrogenase transcript in adult rat tissues.

The mammalian class I alcohol dehydrogenase is the principal enzyme responsible for ethanol metabolism. While it is regarded primarily as a liver-specific enzyme, class I alcohol dehydrogenase is known to be present in a number of extrahepatic tissues. The purpose of the current study is to define the tissue and cellular distribution of the dehydrogenase transcript in four rat tissues previously shown to contain high levels of mRNA: the liver, the proximal small intestine, the colon and the testis. Localization of the transcript was examined in formalin-fixed, paraffin-embedded rat tissues by in situ hybridization using radioactively labelled antisense rat alcohol dehydrogenase RNA probe. In the liver, the dehydrogenase message is localized primarily to the perivenous hepatocytes. In the proximal small intestine and the colon, the message follows a vertical gradient of distribution along the crypt-villus and the crypt-surface epithelium axes, respectively, with the base of the crypt exhibiting the greatest concentration. In the testis, the message is localized primarily to cells in the interstitium. These findings illustrate a highly compartmentalized nature of distribution of the class I alcohol dehydrogenase transcript in the tissues studied and may help to elucidate the metabolic functions of this enzyme in these tissues.

Expression of the class I alcohol dehydrogenase gene in developing rat fetuses.

Class I alcohol dehydrogenase (ADH) is the principal enzyme responsible for ethanol oxidation in mammals. Although primarily regarded as an enzyme that functions in the adult, Class I ADH has been reported to be present in fetal tissues. By in situ hybridization, we demonstrated the tissue localization of the Class I ADH transcript in developing rat fetuses between Days 15 (E15) and 18 (E18) of gestation. Abundant transcripts were present in epidermis, lung, and urinary bladder. In these tissues, the messages were localized primarily to the superficial layer of the epithelium and increased with development. The liver exhibited significant signals only in the E18 fetus, when parenchymal hepatocytes first appeared. The E15 and E16 small intestines, with their epithelium arranged in a stratified fashion, displayed signals in the submucosal mesenchymal layer. By E17, a rearrangement of the intestinal epithelium into an almost monolayer configuration was observed. This change was associated with a redistribution of the ADH transcript to the surface of the epithelium. Further relocation of the messages was noted in the adult small intestine, in which they became concentrated in the base of the crypt. These findings indicate that expression of the rat class I ADH gene follows a dynamic course in specific epithelial tissues during fetal development. In addition, the apparent superficial localization of the ADH message in most of these tissues suggests that ADH functions in metabolizing either endogenously or exogenously derived alcohol substrates present in the fetal environment.

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.

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.

Extracellular matrix from embryonic myocardium elicits an early morphogenetic event in cardiac endothelial differentiation.

A critical step in early cardiac morphogenesis can be faithfully duplicated in culture using a hydrated collagen substratum, and thereby serves as a useful model system for studying the molecular mechanisms of cell differentiation. Results from previous work suggested that the myocardium in the atrioventricular canal (AV) region of the developing chick heart secretes extracellular proteins into its associated basement membrane, which may function to promote an epithelial-mesenchymal transition of endothelium to form prevalvular fibroblasts (E. L. Krug, R. B. Runyan, and R. R. Markwald, 1985, Dev. Biol. 112, 414-426; C. H. Mjaatvedt, R. C. Lepera, and R. R. Markwald, 1987, Dev. Biol., in press). In the present study we show that an EDTA-soluble extract of embryonic chick hearts can substitute for the presence of myocardium, the presumptive stimulator tissue, in initiating mesenchyme formation from AV endothelium in culture. Ventricular endothelium was unresponsive to this material in keeping with observed in situ behavior. AV endothelial cells did not survive beyond 4-5 days when cultured in the absence of either the EDTA-soluble heart extract, myocardial conditioned medium, or the myocardium itself. Antibody prepared against a particulate fraction of the EDTA-solubilized heart extract immunohistochemically localized this material to the myocardial basement membrane. In addition, conditioned medium from embryonic myocardial cultures effectively induced mesenchyme formation. Neither a variety of growth factors nor a sarcoma basement membrane preparation were effective in promoting mesenchyme formation indicating a selectivity of the responding embryonic AV endothelial cells to myocardial basement membrane. These observations reflect a truly inductive phenomenon as there was an absolute dependence on the presence of the stimulating substance/tissue and retention, in culture, of both the temporal and regional characteristics observed in situ. This is in contrast to the results of others investigating the cytodifferentiation of committed cells whose phenotypic expression can be either accelerated or diminished but not obligatorily regulated by a specific agent, thus making the interpretation of data difficult, if not irrelevant, to the study of differentiation. The results of this study provide direct experimental support for the hypothesis that extracellular matrix can indeed serve as a direct stimulator or "secondary inducer" of cytodifferentiation.

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.