Convergent proliferative response and divergent morphogenic pathways induced by epicardial and endocardial signaling in fetal heart development

Heart development requires cardiomyocyte proliferation, coupled with morphogenic differentiation of the inner trabecular myocardium and the outer compact zone myocardium. In mouse embryos lacking the retinoic acid receptor RXRalpha, proliferation and morphogenesis of the compact zone fails. We demonstrated previously that epicardial cells, in response to retinoic acid, secrete an activity that promotes cell proliferation. In this study, we have investigated downstream signaling pathways that are elicited in response to this factor. We find that cells treated in culture activate PI3 kinase and Erk pathways, and that these are required for a proliferative response. In vivo, phosphorylation of Akt and GSK3beta (PI3K pathway) and of Erk1/2 and p90rsk (Erk pathway) is substantially reduced in RXRalpha-deficient heart tissue. Neuregulin, a mitogen secreted from the endocardium which promotes cardiomyocyte proliferation and trabecular differentiation, also activates proliferation via PI3K and Erk pathways. However, the epicardial factor is not neuregulin, and does not function via the neuregulin receptor. Gene markers known to be selectively expressed in trabecular or compact myocardium in vivo are differentially activated in cell culture by treatment with neuregulin or epicardial factor, and are misexpressed in RXRalpha(-/-) heart tissue. We therefore conclude that epicardial and endocardial signals converge on common proliferative components, but diverge in downstream pathways that lead to compact vs. trabecular morphogenic differentiation.     

Msx-1 gene expression and regulation in embryonic palatal tissue

Summary The palatal cleft seen in Msx-1 knock-out mice suggests a role for this gene in normal palate development. The cleft is presumed secondary to tooth and jaw malformations, since in situ hybridization suggests that Msx-1 mRNA is not highly expressed in developing palatal tissue. In this study we demonstrate, by Northern blot analysis, the expression of Msx-1, but not Msx-2, in the developing palate and in primary cultures of murine embryonic palate mesenchymal cells. Furthermore, we propose a role for Msx-1 in retinoic acid-induced cleft palate, since retinoic acid inhibits Msx-1 mRNA expression in palate mesenchymal cells. We also demonstrate that transforming growth factor beta inhibits Msx-1 mRNA expression in palate mesenchymal cells, with retinoic acid and transforming growth factor beta acting synergistically when added simultaneously to these cells. These data suggest a mechanistic interaction between retinoic acid, transforming growth factor beta, and Msx-1 in the etiology of retinoic acid-induced cleft palate.     

Retinoic acid signaling is essential for formation of the heart tube in Xenopus

Retinoic acid is clearly important for the development of the heart. In this paper, we provide evidence that retinoic acid is essential for multiple aspects of cardiogenesis in Xenopus by examining embryos that have been exposed to retinoic acid receptor antagonists. Early in cardiogenesis, retinoic acid alters the expression of key genes in the lateral plate mesoderm including Nkx2.5 and HAND1, indicating that early patterning of the lateral plate mesoderm is, in part, controlled by retinoic acid. We found that, in Xenopus, the transition of the heart from a sheet of cells to a tube required retinoic acid signaling. The requirement for retinoic acid signaling was determined to take place during a narrow window of time between embryonic stages 14 and 18, well before heart tube closure. At the highest doses used, the lateral fields of myocardium fail to fuse, intermediate doses lead to a fusion of the two sides but failure to form a tube, and embryos exposed to lower concentrations of antagonist form a heart tube that failed to complete all the landmark changes that characterize looping. The myocardial phenotypes observed when exposed to the retinoic acid antagonist resemble the myocardium from earlier stages of cardiogenesis, although precocious expression of cardiac differentiation markers was not seen. The morphology of individual cells within the myocardium appeared immature, closely resembling the shape and size of cells at earlier stages of development. However, the failures in morphogenesis are not merely a slowing of development because, even when allowed to develop through stage 40, the heart tubes did not close when embryos were exposed to high levels of antagonist. Indeed, some aspects of left-right asymmetry also remained even in hearts that never formed a tube. These results demonstrate that components of the retinoic acid signaling pathway are necessary for the progression of cardiac morphogenesis in Xenopus.     

Differential expression of TGF beta 1, beta 2 and beta 3 genes during mouse embryogenesis

We have examined by Northern analysis and in situ hybridisation the expression of TGF beta 1, beta 2 and beta 3 during mouse embryogenesis. TGF beta 1 is expressed predominantly in the mesodermal components of the embryo e.g. the hematopoietic cells of both fetal liver and the hemopoietic islands of the yolk sac, the mesenchymal tissues of several internal organs and in ossifying bone tissues. The strongest TGF beta 2 signals were found in early facial mesenchyme and in some endodermal and ectodermal epithelial cell layers e.g., lung and cochlea epithelia. TGF beta 3 was strongest in prevertebral tissue, in some mesothelia and in lung epithelia. All three isoforms were expressed in bone tissues but showed distinct patterns of expression both spatially and temporally. In the root sheath of the whisker follicle, TGF beta 1, beta 2 and beta 3 were expressed simultaneously. We discuss the implication of these results in regard to known regulatory elements of the TGF beta genes and their receptors.     

Transforming growth factor β2 is negatively regulated by endogenous retinoic acid during early heart morphogenesis

Vitamin A‐deficient (VAD) quail embryos lack the vitamin A‐active form, retinoic acid (RA) and are characterized by a phenotype that includes a grossly abnormal cardiovascular system that can be rescued by RA. Here we report that the transforming growth factor, TGFβ2 is involved in RA‐regulated cardiovascular development. In VAD embryos TGFβ2 mRNA and protein expression are greatly elevated. The expression of TGFβ receptor II is also elevated in VAD embryos but is normalized by treatment with TGFβ2‐specific antisense oligonucleotides (AS). Administration of this AS or an antibody specific for TGFβ2 to VAD embryos normalizes posterior heart development and vascularization, while the administration of exogenous active TGFβ2 protein to normal quail embryos mimics the excessive TGFβ2 status of VAD embryos and induces VAD cardiovascular phenotype. In VAD embryos pSmad2/3 and pErk1 are not activated, while pErk2 and pcRaf are elevated and pSmad1/5/8 is diminished. We conclude that in the early avian embryo TGFβ2 has a major role in the retinoic acid‐regulated posterior heart morphogenesis for which it does not use Smad2/3 pathways, but may use other signaling pathways. Importantly, we conclude that retinoic acid is a critical negative physiological regulator of the magnitude of TGFβ2 signals during vertebrate heart formation.     

Complex regulation of TGF beta expression by retinoic acid in the vitamin A-deficient rat

We report the results of a histochemical study, using polyclonal antipeptide antibodies to the different TGF beta isoforms, which demonstrates that retinoic acid regulates the expression of TGF beta 2 in the vitamin A-deficient rat. Basal expression of TGF beta 2 diminished under conditions of vitamin A deficiency. Treatment with retinoic acid caused a rapid and transient induction of TGF beta 2 and TGF beta 3 in the epidermis, tracheobronchial and alveolar epithelium, and intestinal mucosa. Induction of TGF beta 1 expression was also observed in the epidermis. In contrast to these epithelia, expression of the three TGF beta isoforms increased in vaginal epithelium during vitamin A deficiency, and decreased following systemic administration of retinoic acid. Our results show for the first time the widespread regulation of TGF beta expression by retinoic acid in vivo, and suggest a possible mechanism by which retinoics regulate the functions of both normal and pre-neoplastic epithelia.     

Quantification and localization of expression of the retinoic acid receptor-beta and -gamma mRNA isoforms during neurulation in mouse embryos with or without spina bifida

BACKGROUND: Previous studies observed that retinoic acid receptor-gamma (RARgamma) is expressed in the open caudal neuroepithelium but that RARbeta is expressed in the closed neural tube. Furthermore, retinoic acid (RA) induces RARbeta expression, a molecular event associated with neural tube closure, but treatment with RA at the appropriate gestation time causes failure of neural tube closure. Since there are four isoforms of RARbeta, perhaps the isoforms expressed in the closed neural tube and induced by RA are different. To investigate the hypothesis that the switch from RARgamma to RARbeta is mechanistically linked to neural tube closure, this study determined the concentrations and distributions of RARbeta and RARgamma isoforms in mouse embryos with RA-induced neural tube defects and in splotch (Sp) mutant embryos with spina bifida. METHODS: Absolute concentrations of RARbeta and RARgamma isoforms were determined throughout primary neurulation (gestational day 8.5-10.0) in treated or untreated C57BL/6J mouse whole embryos by ribonuclease protection analysis. Treatment consisted of an oral dose of 100 mg/kg of all-trans-RA on gestational day 8.5. Spatial distributions of RARbeta and RARgamma were examined in RA-treated and Sp mutant embryos by in situ hybridization. RESULTS: RARbeta2, gamma1, and gamma2 were expressed in untreated embryos and were induced 4.5-, 1.6-, and 4.0-fold, respectively, 4 hr after treatment with RA. In embryos with RA-induced spina bifida, RARbeta2 was expressed in the closed neural tube while RARgamma1 and RARgamma2 were expressed in the open caudal neuroepithelium. In splotch mice with spina bifida, the boundary between RARbeta and RARgamma did not correspond to the site of neural tube closure. CONCLUSIONS: In RA-treated embryos, the relationship between RARbeta expression in the closed and RARgamma in the open caudal neuroepithelium was not altered. However, in splotch embryos with spina bifida, the juncture between RARbeta and RARgamma expression remained in the same anatomical position in the neuroepithelium irrespective of the neural tube closure status and suggests that the switch from RARgamma to RARbeta expression in the closing caudal neuroepithelium may not be causally linked to neural tube closure in the splotch mutant.     

TGF beta in murine morphogenetic processes: the early embryo and cardiogenesis.

The tissue distribution of TGF beta-1 RNA was examined within whole mouse embryos from implantation to 10.5 days gestational age and, in the developing heart, up to 8 days postpartum. The earliest high level expression of TGF beta-1 RNA is at 7.0 days postcoitum (p.c.) in the cardiac mesoderm. At 8.0 days gestational age, cardiac TGF beta-1 RNA expression is limited to endocardial cells. By 9.5 days p.c., this expression pattern becomes regionalized to those cells that overlie cardiac cushion tissue. High TGF beta-1 RNA levels continue to persist in endothelial cells of the heart valves until approximately one week postpartum. The TGF beta-1 RNA distribution was compared with the extracellular distributions of polypeptides for TGF beta and J1/tenascin. As previously reported, endothelial expression of TGF beta-1 RNA is correlated with mesenchymal expression of TGF beta polypeptide, suggesting a paracrine mode of action for this growth factor in cardiac development. Minor discrepancies in the distributions of TGF beta-1 RNA and the extracellular form of the TGF beta polypeptide suggest that translational or post-translational control of protein levels occurs and/or the possibility that the antibody used may also recognise other members of the TGF beta polypeptide family. A correlation between endothelial TGF beta-1 expression and distribution of J1/tenascin in the mesenchyme gives further support to the proposition that the biological effects of TGF beta-1 may, in part, be mediated by J1/tenascin.     

Dynamic changes in endoglin expression in the developing mouse heart

Endoglin (ENG) is essential for cardiovascular development and is expressed in the heart from its earliest developmental stages. ENG expression has been reported in the cardiac crescent, endocardium, valve mesenchyme and coronary vascular endothelial cells. However, its expression in these cell types is non-uniform and the dynamic changes in ENG expression during heart development have not been systematically studied.Using immunofluorescent staining we tracked ENG protein expression in mouse embryonic hearts aged from 11.5 to 17.5 days, and in postnatal and adult hearts. ENG is expressed in the endocardium and in venous endothelial cells throughout these developmental stages. ENG protein is down-regulated by approximately two-fold as a subset of early coronary veins reprogram to form arteries within the developing myocardium from E13.5. This two-fold higher ratio of ENG protein in veins versus arteries is maintained throughout cardiac development and in the adult heart.ENG is also down-regulated two-fold following mesenchymal transition of endocardial cells to form cardiac valve mesenchyme, whilst expression of the pan-endothelial marker CD31 is completely lost. A subset of epicardial cells (which do not express ENG protein) delaminate and undergo a similar mesenchymal transition to form epicardially derived cells (EPDCs). This transient intra-myocardial mesenchymal cell population expresses low levels of ENG protein, similar to valve mesenchyme.In conclusion, ENG shows dynamic changes of expression in vascular endothelial cells, endocardial cells and mesenchymal cells in the developing heart that vary according to cardiovascular cell type.     

Antisense knockdown of the beta1 integrin subunit in the chicken embryo results in abnormal neural crest cell development

Neural crest cells escape the neural tube by undergoing an epithelial to mesenchymal transition (EMT). This is followed by extensive migration along specific pathways that are lined with extracellular matrix (ECM). In this study, we have examined the roles of matrix receptors containing beta1 integrin subunits in neural crest cell morphogenesis using antisense morpholino oligos electroporated in ovo into avian neural crest cell precursors. Our results show that reduced levels of expression of beta1 integrin subunits in the dorsal neural tube results in an abnormal epithelial to mesenchymal transition. In approximately half of the experimental embryos, however, some neural crest cells filled with beta1 antisense are able to escape the neural tube and migrate ventrally, indicating that grossly normal migration of trunk neural crest cells can take place after beta1 integrin expression is reduced. This study shows the potential of this novel method for investigating the roles of genes that are required for the survival of early mouse embryos in later development events.     

Epithelial-mesenchymal cell transformation in the embryonic heart can be mediated, in part, by transforming growth factor beta

Progenitor cells of the valves and membranous septa of the vertebrate heart are formed by transformation of a specific population of endothelial cells into mesenchyme. Previous studies have shown that this epithelial-mesenchymal cell transformation is mediated by a signal produced by the myocardium of the atrioventricular (AV) canal and transferred across the extracellular matrix. Data are presented here that transforming growth factor beta (TGF beta 1 or TGF beta 2), in combination with an explant of ventricular myocardium, will produce an epithelial-mesenchymal transformation by cultured AV canal endothelial cells in vitro. Alone, neither component is capable of producing this effect. The factor provided by the ventricular explant cannot be substituted by either epidermal growth factor or basic fibroblast growth factor. Further experiments show that an antibody that blocks TGF beta activity is effective in preventing the epithelial-mesenchymal cell transformation normally produced by AV canal myocardium. Control antibodies are without effect. By immunological criteria, a member of the TGF beta family of molecules can be demonstrated in the chicken embryo and heart at the time overt valvular formation begins. Together, these data show that TGF beta 1 can produce mesenchymal cell formation in vitro and provide evidence that a member of the TGF beta family is present and plays a role in the process of epithelial-mesenchymal cell transformation in the embryonic heart.     

Endocardial to Myocardial Notch-Wnt-Bmp Axis Regulates Early Heart Valve Development

Endocardial to mesenchymal transformation (EMT) is a fundamental cellular process required for heart valve formation. Notch, Wnt and Bmp pathways are known to regulate this process. To further address how these pathways coordinate in the process, we specifically disrupted Notch1 or Jagged1 in the endocardium of mouse embryonic hearts and showed that Jagged1-Notch1 signaling in the endocardium is essential for EMT and early valvular cushion formation. qPCR and RNA in situ hybridization assays reveal that endocardial Jagged1-Notch1 signaling regulates Wnt4 expression in the atrioventricular canal (AVC) endocardium and Bmp2 in the AVC myocardium. Whole embryo cultures treated with Wnt4 or Wnt inhibitory factor 1 (Wif1) show that Bmp2 expression in the AVC myocardium is dependent on Wnt activity; Wnt4 also reinstates Bmp2 expression in the AVC myocardium of endocardial Notch1 null embryos. Furthermore, while both Wnt4 and Bmp2 rescue the defective EMT resulting from Notch inhibition, Wnt4 requires Bmp for its action. These results demonstrate that Jagged1-Notch1 signaling in endocardial cells induces the expression of Wnt4, which subsequently acts as a paracrine factor to upregulate Bmp2 expression in the adjacent AVC myocardium to signal EMT.     

Expression of transforming growth factor beta 2 RNA during murine embryogenesis

We have studied the temporal and spatial expression of transforming growth factor beta 2 (TGF beta 2) RNA in mouse embryos from 10.5 days post coitum (p.c.) to 3 days post partum (p.p.) by in situ hybridization analysis. TGF beta 2 RNA is expressed in a variety of tissues including bone, cartilage, tendon, gut, blood vessels, skin and fetal placenta, and is in general found in the mesenchymal component of these tissues. The expression of TGF beta 2 RNA changes during development in a manner consistent with a role for the gene product in mediating mesenchymal-epithelial interactions.     

MesP1 is expressed in the heart precursor cells and required for the formation of a single heart tube.

The Mesp1 gene encodes the basic HLH protein MesP1 which is expressed in the mesodermal cell lineage during early gastrulation. Disruption of the Mesp1 gene leads to aberrant heart morphogenesis, resulting in cardia bifida. In order to study the defects in Mesp1-expressing cells during gastrulation and in the specification of mesodermal cell lineages, we introduced a (beta)-galactosidase gene (lacZ) under the control of the Mesp1 promoter by homologous recombination. The early expression pattern revealed by (beta)-gal staining in heterozygous embryos was almost identical to that observed by whole mount in situ hybridization. However, the (beta)-gal activity was retained longer than the mRNA signal, which enabled us to follow cell migration during gastrulation. In heterozygous embryos, the Mesp1-expressing cells migrated out from the primitive streak and were incorporated into the head mesenchyme and heart field. In contrast, Mesp1-expressing cells in the homozygous deficient embryos stayed in the primitive streak for a longer period of time before departure. The expression of FLK-1, an early marker of endothelial cell precursors including heart precursors, also accumulated abnormally in the posterior region in Mesp1-deficient embryos. In addition, using the Cre-loxP site-specific recombination system, we could determine the lineage of the Mesp1-expressing cells. The first mesodermal cells that ingressed through the primitive streak were incorporated as the mesodermal component of the amnion, and the next mesodermal population mainly contributed to the myocardium of the heart tube but not to the endocardium. These results strongly suggest that MesP1 is expressed in the heart tube precursor cells and is required for mesodermal cells to depart from the primitive streak and to generate a single heart tube.     

Expression of vinexin alpha in the dorsal half of the eye and in the cardiac outflow tract and atrioventricular canal

The Smads are intracellular signalling molecules that transduce signals from receptors for members of the TGF-beta superfamily to the nucleus. We have cloned the Xenopus orthologue of Smad3 (XSmad3). It is 94.6% identical to human Smad3 at the amino acid level. It is expressed as a maternal mRNA which disappears after stage 10.5, but reappears at the early tailbud stages. It is much less abundant than XSmad2 at the early developmental stages. From Stage 27 onwards XSmad3 is expressed with XSmad2 throughout the head region and in the somitic region. Strikingly however, XSmad3 alone is specifically expressed in the chordoneural hinge, the notochord and in the developing heart. Closer analysis reveals that XSmad3 is specifically expressed in the endocardium but not in the myocardium or pericardium. The chordoneural hinge staining persists at least until stage 40 whereas the staining in the endocardium peaks at approximately stage 32/33.     

Expression of activins and TGF beta 1 and beta 2 RNAs in early postimplantation mouse embryos and uterine decidua.

The expression of the mesoderm inducing factors, activins and TGF beta s, was characterized in 5 1/2-9 1/2 day mouse embryos and implantation sites by in situ hybridization. Activin beta A RNA was not detected within the embryo, but is expressed in nearby decidual cells from 5 to 7 days. Thus activin A could play a role within the embyro during gastrulation. Activin beta A is also expressed in more mesometrially located decidual cells from 6 to 9 1/2 days. Activin beta B and inhibin alpha RNAs were not detected, while a control tissue was highly positive. TGF beta 1 is expressed in the secondary decidual zone and in developing endothelial cells in the decidua and embryo. TGF beta 2 is expressed in the mesometrial decidua at 6 1/2 days and in the midline of the cranial neural plate.