Comparison of the four mouse fasciclin-containing genes expression patterns during valvuloseptal morphogenesis

All four mammalian fasciclin-containing genes are expressed in the adult valves and are localized in partially overlapping and reciprocal patterns during cardiovascular development. Spatiotemporal comparison of the fasciclin-containing secreted adhesion genes, TGFbeta induced clone H3 (betaigH3) and periostin, revealed that they are co-localized within the outflow tract endocardial cushions, but that betaigH3 expression is restricted to the septal cushions within the atrioventricular canal. Conversely, the fasciclin-containing transmembrane gene, stabilin-1, is predominately expressed in the endocardial layer overlaying the cushions and lining the developing heart. However, expression of the fasciclin-containing transmembrane gene, stabilin-2 is only present in the post-natal mature valve endothelial cells. These data illustrate for the first time that the primitive endocardial cushions dynamically express multiple fasciclin-containing adhesion molecules as they undergo the key steps of seeding, proliferation, differentiation, fusion, mesenchymal condensation and remodeling during mouse heart development.     

Temporal and distinct TGFbeta ligand requirements during mouse and avian endocardial cushion morphogenesis

The formation of endocardial cushions in the atrioventricular (AV) canal of the rudimentary heart requires epithelial-to-mesenchymal cell transformation (EMT). This is a complex developmental process regulated by multiple extracellular signals and transduction pathways. A collagen gel assay, long used to examine endocardial cushion development in avian models, is now being employed to investigate genetically engineered mouse models with abnormal heart morphogenesis. In this study, we determine interspecies variations for avian and mouse cultured endocardial cushion explants. Considering these observed morphologic differences, we also define the temporal requirements for TGFbeta2 and TGFbeta3 during mouse endocardial cushion morphogenesis. TGFbeta2 and TGFbeta3 blocking antibodies inhibit endothelial cell activation and transformation, respectively, in avian explants. In contrast, neutralizing TGFbeta2 inhibits cell transformation in the mouse, while TGFbeta3 antibodies have no effect on activation or transformation events. This functional requirement for TGFbeta2 is concomitant with expression of TGFbeta2, but not TGFbeta3, within mouse endocardial cushions at a time coincident with transformation. Thus, both TGFbeta2 and TGFbeta3 appear necessary for the full morphogenetic program of EMT in the chick, but only TGFbeta2 is expressed and obligatory for mammalian endocardial cushion cell transformation.     

Loss of muscleblind-like 1 promotes invasive mesenchyme formation in endocardial cushions by stimulating autocrine TGFbeta3

ABSTRACT: BACKGROUND: Valvulogenesis and septation in the developing heart depend on the formation and remodeling of endocardial cushions in the atrioventricular canal (AVC) and outflow tract (OFT). These cushions are invaded by a subpopulation of endocardial cells that undergo an epithelial-mesenchymal transition in response to paracrine and autocrine transforming growth factor beta (TGFbeta) signals. We previously demonstrated that the RNA binding protein muscleblind-like 1 (MBNL1) is expressed specifically in the cushion endocardium, and knockdown of MBNL1 in stage 14 embryonic chicken AVC explants enhances TGFbeta-dependent endocardial cell invasion. RESULTS: In this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGFbeta3 after it is no longer required to induce basal levels of invasion. TGFbeta3, but not TGFbeta2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGFbeta3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGFbeta3 production in the endocardium. More TGFbeta3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGFbeta3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGFbeta3 secretion, and early exposure to excess TGFbeta3 induces precocious invasion. MBNL1 expression precedes TGFbeta3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGFbeta3 signaling. The stimulatory effects of MBNL1 knockdown on invasion are lost in stage 16 AVC explants. Knockdown of MBNL1 in OFT explants similarly enhances cell invasion, but not activation. TGFbeta is necessary and sufficient to mediate this effect. CONCLUSIONS: Taken together, these data support a model in which MBNL1 negatively regulates cell invasion in the endocardial cushions by restricting the magnitude and timing of endocardial-derived TGFbeta3 production.     

Periostin expression by epicardium-derived cells is involved in the development of the atrioventricular valves and fibrous heart skeleton

Abstract The epicardium is embryologically formed by outgrowth of proepicardial cells over the naked heart tube. Epicardium-derived cells (EPDCs) migrate into the myocardium, contributing to myocardial architecture, valve development, and the coronary vasculature. Defective EPDC formation causes valve malformations, myocardial thinning, and coronary defects. In the atrioventricular (AV) valves and the fibrous heart skeleton isolating atrial from ventricular myocardium, EPDCs colocalize with periostin, a matrix molecule involved in remodeling. We investigated whether proepicardial outgrowth inhibition affected periostin expression and how this related to development of the AV valves and fibrous heart skeleton.
Periostin expression by epicardium and EPDCs was confirmed in vitro in primary cultures of human and quail EPDCs. Disturbing EPDC formation in quail embryos reduced periostin expression in the endocardial cushions and AV junction. Disturbed fibrous tissue development resulted in AV myocardial connections reflected by preexcitation electrocardiographic patterns.
We conclude that EPDCs are local producers of periostin. Disturbance of EPDC formation results in decreased cardiac periostin levels and hampers the development of fibrous tissue in AV junction and the developing AV valves. The resulting cardiac anomalies might link to Wolff–Parkinson White syndrome with persistent AV myocardial connections.     

Signal transduction in early heart development (II): ventricular chamber specification, trabeculation, and heart valve formation

The formation of a four-chambered heart with ventricular chambers aligned in a left-right orientation begins with the rightward looping of the linear heart tube in accordance with the left-right embryonic axis. The functional specification of the ventricular chambers in the looped heart occurs with the formation of a trabeculated myocardium along the outer curvature of the realigned heart tube. Two major signal transduction pathways are involved in this process, the retinoic acid and neuregulin signaling pathways, with the retinoic acid pathway also participating in rightward heart tube looping. With the establishment of the atrial and ventricular chambers, maintenance of a unidirectional flow of blood between the two chambers must be ensured. To achieve this, heart valves develop at the atrioventricular juncture. This process begins with formation of endocardial cushions, the primordia of heart valves, and ends with formation of heart valve leaflets. Underlying this process is a complex network of signal transduction pathways that mediate communication between the endocardial and myocardial cell layers to form the endocardial cushions and nascent heart valve. Some of the signaling molecules involved are vascular endothelial growth factor, Wnts, bone morphogenetic proteins, epidermal growth factor, hyaluronic acid, neurofibromin, and calcium.     

Functional BMP receptor in endocardial cells is required in atrioventricular cushion mesenchymal cell formation in chick

Transformation of atrioventricular (AV) canal endocardium into invasive mesenchyme correlates spatially and temporally with the expression of bone morphogenetic protein (BMP)-2 in the AV myocardium. We revealed the presence of mRNA of Type I BMP receptors, BMPR-1A (ALK3), BMPR-1B (ALK6) and ALK2 in chick AV endocardium at stage-14(-), the onset of epithelial to mesenchymal transformation (EMT), by RT-PCR and localized BMPR-1B mRNA in the endocardium by in situ hybridization. To circumvent the functional redundancies among the Type I BMP receptors, we applied dominant-negative (dn) BMPR-1B-viruses to chick AV explants and whole-chick embryo cultures to specifically block BMP signaling in AV endocardium during EMT. dnBMPR-1B-virus infection of AV endocardial cells abolished BMP-2-supported AV endocardial EMT. Conversely, caBMPR-1B-virus infection promoted AV endocardial EMT in the absence of AV myocardium. Moreover, dnBMPR-1B-virus treatments significantly reduced myocardially supported EMT in AV endocardial-myocardial co-culture. AV cushion mesenchymal cell markers, alpha-smooth muscle actin (SMA), and TGFbeta3 in the endocardial cells were promoted by caBMPR-1B and reduced by dnBMPR-1B infection. Microinjection of the virus into the cardiac jelly in the AV canal at stage-13 in vivo (ovo) revealed that the dnBMPR-1B-virus-infected cells remained in the endocardial epithelium, whereas caBMPR-1B-infected cells invaded deep into the cushions. These results provide evidence that BMP signaling through the AV endocardium is required for the EMT and the activation of the BMP receptor in the endocardium can promote AV EMT in the chick.     

Atrioventricular cushion transformation is mediated by ALK2 in the developing mouse heart

Developmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-beta superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-beta superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-beta Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis.     

A novel role for VEGF in endocardial cushion formation and its potential contribution to congenital heart defects

Normal cardiovascular development is exquisitely dependent on the correct dosage of the angiogenic growth factor and vascular morphogen vascular endothelial growth factor (VEGF). However, cardiac expression of VEGF is also robustly augmented during hypoxic insults, potentially mediating the well-established teratogenic effects of hypoxia on heart development. We report that during normal heart morphogenesis VEGF is specifically upregulated in the atrioventricular (AV) field of the heart tube soon after the onset of endocardial cushion formation (i.e. the endocardium-derived structures that build the heart septa and valves). To model hypoxia-dependent induction of VEGF in vivo, we conditionally induced VEGF expression in the myocardium using a tetracycline-regulated transgenic system. Premature induction of myocardial VEGF in E9.5 embryos to levels comparable with those induced by hypoxia prevented formation of endocardial cushions. When added to explanted embryonic AV tissue, VEGF fully inhibited endocardial-to-mesenchymal transformation. Transformation was also abrogated in AV explants subjected to experimental hypoxia but fully restored in the presence of an inhibitory soluble VEGF receptor 1 chimeric protein. Together, these results suggest a novel developmental role for VEGF as a negative regulator of endocardial-to-mesenchymal transformation that underlies the formation of endocardial cushions. Moreover, ischemia-induced VEGF may be the molecular link between hypoxia and congenital defects in heart septation.     

Expression of cellular retinoic acid-binding protein I and II (CRABP I and II) in embryonic mouse hearts treated with retinoic acid

Cellular retinoic acid binding proteins are considered to be involved in retinoic acid (RA) signaling pathways. Our aim was to compare the expression and localization of cellular retinoic acid binding proteins I and II (CRABP I and II) in embryonic mouse hearts during normal development and after a single teratogenic dose of RA. Techniques such as real-time PCR, RT-PCR, Western blots and immunostaining were employed to examine hearts from embryos at 9-17 dpc. RA treatment at 8.5dpc affects production of CRABP I and II in the heart in the 48-h period. Changes in expression of mRNA for retinaldehyde dehydrogenase II (Raldh2), Crabp1 and Crabp2 genes also occur within the same time window (i.e. 10-11dpc) after RA treatment. In the embryonic control heart these proteins are localized in groups of cells within the outflow tract (OT), and the atrioventricular endocardial cushions. A gradient of labeling is observed with CRABP II but not for CRABP I along the myocardium of the looped heart at 11 dpc; this gradient is abolished in hearts treated with RA, whereas an increase of RALDH2 staining has been observed at 10 dpc in RA-treated hearts. Some populations of endocardial endothelial cells were intensively stained with anti-CRABP II whereas CRABP I was negative in these structures. These results suggest that CRABP I and II are independently regulated during heart development, playing different roles in RA signaling, essential for early remodeling of the heart tube and alignment of the great arteries to their respective ventricles.     

Tbx20 regulation of endocardial cushion cell proliferation and extracellular matrix gene expression

While recent work has implicated Tbx20 in myocardial maturation and proliferation, the role of Tbx20 in heart valve development remains relatively unknown. Tbx20 expression was manipulated in primary avian endocardial cells in order to elucidate its function in developing endocardial cushions. Tbx20 gain of function was achieved with a Tbx20-adenovirus, and endogenous Tbx20 expression was inhibited with Tbx20-specific siRNA in cultured endocardial cushion cells. With Tbx20 gain of function, the expression of chondroitin sulfate proteoglycans (CSPG), including aggrecan and versican, was decreased, while the expression of the matrix metalloproteinases (MMP) mmp9 and mmp13 was increased. Consistent results were observed with Tbx20 loss of function, where the expression of CSPG genes increased and MMP genes decreased. In addition, cushion mesenchyme proliferation increased with infection of a Tbx20-adenovirus and decreased with transfection of Tbx20-specfic siRNA. Furthermore, BMP2 treatment resulted in increased Tbx20 expression in endocardial cushion cells, and loss of Tbx20 led to increased Tbx2 and decreased N-myc gene expression. Taken together, these data support a role for Tbx20 in repressing extracellular matrix remodeling and promoting cell proliferation in mesenchymal valve precursor populations in endocardial cushions during embryonic development.     

Elevated transforming growth factor beta2 enhances apoptosis and contributes to abnormal outflow tract and aortic sac development in retinoic X receptor alpha knockout embryos

Septation of the single tubular embryonic outflow tract into two outlet segments in the heart requires the precise integration of proliferation, differentiation and apoptosis during remodeling. Lack of proper coordination between these processes would result in a variety of congenital cardiac defects such as those seen in the retinoid X receptor alpha knockout (Rxra(-/-)) mouse. Rxra(-/-) embryos exhibit lethality between embryonic day (E) 13.5 and 15.5 and harbor a variety of conotruncal and aortic sac defects making it an excellent system to investigate the molecular and morphogenic causes of these cardiac malformations. At E12.5, before the embryonic lethality, we found no qualitative difference between wild type and Rxra(-/-) proliferation (BrdU incorporation) in outflow tract cushion tissue but a significant increase in apoptosis as assessed by both TUNEL labeling in paraffin sections and caspase activity in trypsin-dispersed hearts. Additionally, E12.5 embryos demonstrated elevated levels of transforming growth factor beta2 (TGFbeta2) protein in multiple cell lineages in the heart. Using a whole-mouse-embryo culture system, wild-type E11.5 embryos treated with TGFbeta2 protein for 24 hours displayed enhanced apoptosis in both the sinistroventralconal cushion and dextrodorsalconal cushion in a manner analogous to that observed in the Rxra(-/-). TGFbeta2 protein treatment also led to malformations in both the outflow tract and aortic sac. Importantly, Rxra(-/-) embryos that were heterozygous for a null mutation in the Tgfb2 allele exhibited a partial restoration of the elevated apoptosis and of the malformations. This was evident at both E12.5 and E13.5. The data suggests that elevated levels of TGFbeta2 can (1) contribute to abnormal outflow tract morphogenesis by enhancing apoptosis in the endocardial cushions and (2) promote aortic sac malformations by interfering with the normal development of the aorticopulmonary septum.     

Thymosin Beta4 Regulates Cardiac Valve Formation Via Endothelial-Mesenchymal Transformation in Zebrafish Embryos

Thymosin beta4 (TB4) has multiple functions in cellular response in processes as diverse as embryonic organ development and the pathogeneses of disease, especially those associated with cardiac coronary vessels. However, the specific roles played by TB4 during heart valve development in vertebrates are largely unknown. Here, we identified a novel function of TB4 in endothelialmesenchymal transformation (EMT) in cardiac valve endocardial cushions in zebrafish. The expressions of thymosin family members in developing zebrafish embryos were determined by whole mount in situ hybridization. Of the thymosin family members only zTB4 was expressed in the developing heart region. Cardiac valve development at 48 h post fertilization was defected in zebrafish TB4 (zTB4) morpholino-injected embryos (morphants). In zTB4 morphants, abnormal linear heart tube development was observed. The expressions of bone morphogenetic protein (BMP) 4, notch1b, and hyaluronic acid synthase (HAS) 2 genes were also markedly reduced in atrio-ventricular canal (AVC). Endocardial cells in the AVC region were stained with anti-Zn5 antibody reactive against Dm-grasp (an EMT marker) to observe EMT in developing cardiac valves in zTB4 morphants. EMT marker expression in valve endothelial cells was confirmed after transfection with TB4 siRNA in the presence of transforming growth factor β (TGFβ) by RT-PCR and immunofluorescent assay. Zn5-positive endocardial AVC cells were not observed in zTB4 morphants, and knockdown of TB4 suppressed TGF-β-induced EMT in ovine valve endothelial cells. Taken together, our results demonstrate that TB4 plays a pivotal role in cardiac valve formation by increasing EMT.     

TGFbeta2 and TGFbeta3 have separate and sequential activities during epithelial-mesenchymal cell transformation in the embryonic heart

Heart valve formation is initiated by an epithelial-mesenchymal cell transformation (EMT) of endothelial cells in the atrioventricular (AV) canal. Mesenchymal cells formed from cardiac EMTs are the initial cellular components of the cardiac cushions and progenitors of valvular and septal fibroblasts. It has been shown that transforming growth factor beta (TGFbeta) mediates EMT in the AV canal, and TGFbeta1 and 2 isoforms are expressed in the mouse heart while TGFbeta 2 and 3 are expressed in the avian heart. Depletion of TGFbeta3 in avian or TGFbeta2 in mouse leads to developmental defects of heart tissue. These observations raise questions as to whether multiple TGFbeta isoforms participate in valve formation. In this study, we examined the localization and function of TGFbeta2 and TGFbeta3 in the chick heart during EMT. TGFbeta2 was present in both endothelium and myocardium before and after EMT. TGFbeta2 antibody inhibited endothelial cell-cell separation. In contrast, TGFbeta3 was present only in the myocardium before EMT and was in the endothelium at the initiation of EMT. TGFbeta3 antibodies inhibited mesenchymal cell formation and migration into the underlying matrix. Both TGFbeta2 and 3 increased fibrillin 2 expression. However, only TGFbeta2 treatment increased cell surface beta-1,4-galactosyltransferase expression. These data suggest that TGFbeta2 and TGFbeta3 are sequentially and separately involved in the process of EMT. TGFbeta2 mediates initial endothelial cell-cell separation while TGFbeta3 is required for the cell morphological change that enables the migration of cells into the underlying ECM.     

Hearts and bones: shared regulatory mechanisms in heart valve, cartilage, tendon, and bone development

The atrioventricular heart valve leaflets and chordae tendineae are composed of diverse cell lineages and highly organized extracellular matrices that share characteristics with cartilage and tendon cell types in the limb buds and somites. During embryonic chicken valvulogenesis, aggrecan and sox9, characteristic of cartilage cells, are observed in the AV valve leaflets, in contrast to tendon-associated genes scleraxis and tenascin, present in the chordae tendineae. In the limb buds and somites, cartilage cell lineage differentiation is regulated by BMP2, while FGF4 controls tendon cell fate. The ability of BMP2 and FGF4 to induce similar patterns of gene expression in heart valve precursor cells was examined. In multiple assays of cells from prefused endocardial cushions, BMP2 is sufficient to activate Smad1/5/8 phosphorylation and induce sox9 and aggrecan expression, while FGF4 treatment increases phosphorylated MAPK (dpERK) signaling and promotes expression of scleraxis and tenascin. However, these treatments do not alter differentiated lineage gene expression in valve progenitors from fused cushions of older embryos. Together, these studies define regulatory pathways of AV valve progenitor cell diversification into leaflets and chordae tendineae that share inductive interactions and differentiation phenotypes with cartilage and tendon cell lineages.