Nephronectin regulates atrioventricular canal differentiation via Bmp4-Has2 signaling in zebrafish

The extracellular matrix is crucial for organogenesis. It is a complex and dynamic component that regulates cell behavior by modulating the activity, bioavailability and presentation of growth factors to cell surface receptors. Here, we determined the role of the extracellular matrix protein Nephronectin (Npnt) in heart development using the zebrafish model system. The vertebrate heart is formed as a linear tube in which myocardium and endocardium are separated by a layer of extracellular matrix termed the cardiac jelly. During heart development, the cardiac jelly swells at the atrioventricular (AV) canal, which precedes valve formation. Here, we show that Npnt expression correlates with this process. Morpholino-mediated knockdown of Npnt prevents proper valve leaflet formation and trabeculation and results in greater than 85% lethality at 7 days post-fertilization. The earliest observed phenotype is an extended tube-like structure at the AV boundary. In addition, the expression of myocardial genes involved in cardiac valve formation (cspg2, fibulin 1, tbx2b, bmp4) is expanded and endocardial cells along the extended tube-like structure exhibit characteristics of AV cells (has2, notch1b and Alcam expression, cuboidal cell shape). Inhibition of has2 in npnt morphants rescues the endocardial, but not the myocardial, expansion. By contrast, reduction of BMP signaling in npnt morphants reduces the ectopic expression of myocardial and endocardial AV markers. Taken together, our results identify Npnt as a novel upstream regulator of Bmp4-Has2 signaling that plays a crucial role in AV canal differentiation.     

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.     

Msx1creERT2 knock‐In allele: A useful tool to target embryonic and adult cardiac valves

Summary : Heart valve development begins with the endothelial‐to‐mesenchymal transition (EMT) of endocardial cells. Although lineage studies have demonstrated contributions from cardiac neural crest and epicardium to semilunar and atrioventricular (AV) valve formation, respectively, most valve mesenchyme derives from the endocardial EMT. Specific Cre mouse lines for fate‐mapping analyses of valve endocardial cells are limited. Msx1 displayed expression in AV canal endocardium and cushion mesenchyme between E9.5 and E11.5, when EMT is underway. Additionally, previous studies have demonstrated that deletion of Msx1 and its paralog Msx2 results in hypoplastic AV cushions and impaired endocardial signaling. A knock‐in tamoxifen‐inducible Cre line was recently generated (Msx1^CreERT2) and characterized during embryonic development and after birth, and was shown to recapitulate the endogenous Msx1 expression pattern. Here, we further analyze this knock‐in allele and track the Msx1‐expressing cells and their descendants during cardiac development with a particular focus on their contribution to the valves and their precursors. Thus, Msx1^CreERT2 mice represent a useful model for lineage tracing and conditional gene manipulation of endocardial and mesenchymal cushion cells essential to understand mechanisms of valve development and remodeling. genesis 53:337–345, 2015. © 2015 Wiley Periodicals, Inc.     

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.     

Bone morphogenetic protein-2 can mediate myocardial regulation of atrioventricular cushion mesenchymal cell formation in mice

Transformation of endocardial endothelial cells into invasive mesenchyme is a critical antecedent of cardiac cushion tissue formation. The message for bone morphogenetic protein (BMP)-2 is known to be expressed in myocardial cells in a manner consistent with the segmental pattern of cushion formation [Development 109(1990) 833]. In the present work, we localized BMP-2 protein in atrioventricular (AV) myocardium in mice at embryonic day (ED) 8.5 (12 somite stage) before the onset of AV mesenchymal cell formation at ED 9.5. BMP-2 protein expression was absent from ventricular myocardium throughout the stages examined. After cellularization of the AV cushion at ED 10.5, myocardial BMP-2 protein expression was diminished in AV myocardium, whereas cushion mesenchymal cells started expressing BMP protein. Expression of BMP-2 in cushion mesenchyme persisted during later stages of development, ED 13.5-16, during valuvulogenesis. Intense expression of BMP-2 persisted in the valve tissue in adult mice. Based on the expression pattern, we performed a series of experiments to test the hypothesis that BMP-2 mediates myocardial regulation of cardiac cushion tissue formation in mice. When BMP-2 protein was added to the 16-18 somite stage (ED 9.25) AV endocardial endothelium in culture, cushion mesenchymal cells were formed in the absence of AV myocardium, which invaded into collagen gels and expressed the mesenchymal marker, smooth muscle (SM) alpha-actin; whereas the endothelial marker, PECAM-1, was lost from the invaded cells. In contrast, when noggin, a specific antagonist to BMPs, was applied together with BMP-2 to the culture medium, AV endothelial cells remained as an epithelial monolayer with little expression of SM alpha-actin, and expression of PECAM-1 was retained in the endocardial cells. When noggin was added to AV endothelial cells cocultured with associated myocardium, it blocked endothelial transformation to mesenchyme. AV endothelium treated with BMP-2 expressed elevated levels of TGFbeta-2 in the absence of myocardium, as observed in the endothelium cocultured with myocardium. BMP-2-supported elevation of TGFbeta-2 expression in endocardial cells was abolished by noggin treatment. These data indicated that BMP signaling is required in and BMP-2 is sufficient for myocardial segmental regulation of AV endocardial cushion mesenchymal cell formation in mice.     

Elevated glucose inhibits VEGF-A-mediated endocardial cushion formation: modulation by PECAM-1 and MMP-2

Atrioventricular (AV) septal defects resulting from aberrant endocardial cushion (EC) formation are observed at increased rates in infants of diabetic mothers. EC formation occurs via an epithelial-mesenchymal transformation (EMT), involving transformation of endocardial cells into mesenchymal cells, migration, and invasion into extracellular matrix. Here, we report that elevated glucose inhibits EMT by reducing myocardial vascular endothelial growth factor A (VEGF-A). This effect is reversed with exogenous recombinant mouse VEGF-A165, whereas addition of soluble VEGF receptor-1 blocks EMT. We show that disruption of EMT is associated with persistence of platelet endothelial cell adhesion molecule-1 (PECAM-1) and decreased matrix metalloproteinase-2 (MMP-2) expression. These findings correlate with retention of a nontransformed endocardial sheet and lack of invasion. The MMP inhibitor GM6001 blocks invasion, whereas explants from PECAM-1 deficient mice exhibit MMP-2 induction and normal EMT in high glucose. PECAM-1-negative endothelial cells are highly motile and express more MMP-2 than do PECAM-1-positive endothelial cells. During EMT, loss of PECAM-1 similarly promotes single cell motility and MMP-2 expression. Our findings suggest that high glucose-induced inhibition of AV cushion morphogenesis results from decreased myocardial VEGF-A expression and is, in part, mediated by persistent endocardial cell PECAM-1 expression and failure to up-regulate MMP-2 expression.     

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.     

Activin receptor-like kinase 2 and Smad6 regulate epithelial-mesenchymal transformation during cardiac valve formation

Epithelial-mesenchymal transformation (EMT) occurs during both development and tumorigenesis. Transforming growth factor beta (TGFbeta) ligands signal EMT in the atrioventricular (AV) cushion of the developing heart, a critical step in valve formation. TGFbeta signals through a complex of type I and type II receptors. Several type I receptors exist although activin receptor-like kinase (ALK) 5 mediates the majority of TGFbeta signaling. Here, we demonstrate that ALK2 is sufficient to induce EMT in the heart. Both ALK2 and ALK5 are expressed throughout the heart with ALK2 expressed abundantly in endocardial cells of the outflow tract (OFT), ventricle, and AV cushion. Misexpression of constitutively active (ca) ALK2 in non-transforming ventricular endocardial cells induced EMT, while caALK5 did not, thus demonstrating that ALK2 activity alone is sufficient to stimulate EMT. Smad6, an inhibitor of Smad signaling downstream of ALK2, but not ALK5, inhibited EMT in AV cushion endocardial cells. These data suggest that ALK2 activation may stimulate EMT in the AV cushion and that Smad6 may act downstream of ALK2 to negatively regulate EMT.     

<Emphasis Type="Italic">Msx1</Emphasis> and <Emphasis Type="Italic">Msx2</Emphasis>are required for endothelial-mesenchymal transformation of the atrioventricular cushions and patterning of the atrioventricular myocardium

Background  

Msx1 and Msx2, which belong to the highly conserved Nk family of homeobox genes, display overlapping expression patterns and redundant functions in multiple tissues and organs during vertebrate development. Msx1 and Msx2 have well-documented roles in mediating epithelial-mesenchymal interactions during organogenesis. Given that both Msx1 and Msx2 are crucial downstream effectors of Bmp signaling, we investigated whether Msx1 and Msx2 are required for the Bmp-induced endothelial-mesenchymal transformation (EMT) during atrioventricular (AV) valve formation.     

Activation of Notch1 signaling in cardiogenic mesoderm induces abnormal heart morphogenesis in mouse

Notch signaling is implicated in many developmental processes. In our current study, we have employed a transgenic strategy to investigate the role of Notch signaling during cardiac development in the mouse. Cre recombinase-mediated Notch1 (NICD1) activation in the mesodermal cell lineage leads to abnormal heart morphogenesis, which is characterized by deformities of the ventricles and atrioventricular (AV) canal. The major defects observed include impaired ventricular myocardial differentiation, the ectopic appearance of cell masses in the AV cushion, the right-shifted interventricular septum (IVS) and impaired myocardium of the AV canal. However, the fates of the endocardium and myocardium were not disrupted in NICD1-activated hearts. One of the Notch target genes, Hesr1, was found to be strongly induced in both the ventricle and the AV canal of NICD1-activated hearts. However, a knockout of the Hesr1 gene from NICD-activated hearts rescues only the abnormality of the AV myocardium. We searched for additional possible targets of NICD1 activation by GeneChip analysis and found that Wnt2, Bmp6, jagged 1 and Tnni2 are strongly upregulated in NICD1-activated hearts, and that the activation of these genes was also observed in the absence of Hesr1. Our present study thus indicates that the Notch1 signaling pathway plays a suppressive role both in AV myocardial differentiation and the maturation of the ventricular myocardium.     

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.     

Hesr1 and Hesr2 regulate atrioventricular boundary formation in the developing heart through the repression of Tbx2

The establishment of chamber specificity is an essential requirement for cardiac morphogenesis and function. Hesr1 (Hey1) and Hesr2 (Hey2) are specifically expressed in the atrium and ventricle, respectively, implicating these genes in chamber specification. In our current study, we show that the forced expression of Hesr1 or Hesr2 in the entire cardiac lineage of the mouse results in the reduction or loss of the atrioventricular (AV) canal. In the Hesr1-misexpressing heart, the boundaries of the AV canal are poorly defined, and the expression levels of specific markers of the AV myocardium, Bmp2 and Tbx2, are either very weak or undetectable. More potent effects were observed in Hesr2-misexpressing embryos, in which the AV canal appears to be absent entirely. These data suggest that Hesr1 and Hesr2 may prevent cells from expressing the AV canal-specific genes that lead to the precise formation of the AV boundary. Our findings suggest that Tbx2 expression might be directly suppressed by Hesr1 and Hesr2. Furthermore, we find that the expression of Hesr1 and Hesr2 is independent of Notch2 signaling. Taken together, our data demonstrate that Hesr1 and Hesr2 play crucial roles in AV boundary formation through the suppression of Tbx2.     

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.Download video file.^{(86M, mov)}     

Zebrafish Crip2 Plays a Critical Role in Atrioventricular Valve Development by Downregulating the Expression of ECM Genes in the Endocardial Cushion

The initial step of atrioventricular (AV) valve development involves the deposition of extracellular matrix (ECM) components of the endocardial cushion and the endocardialmesenchymal transition. While the appropriately regulated expression of the major ECM components, Versican and Hyaluronan, that form the endocardial cushion is important for heart valve development, the underlying mechanism that regulates ECM gene expression remains unclear. We found that zebrafish crip2 expression is restricted to a subset of cells in the AV canal (AVC) endocardium at 55 hours post-fertilization (hpf). Knockdown of crip2 induced a heart-looping defect in zebrafish embryos, although the development of cardiac chambers appeared to be normal. In the AVC of Crip2-deficient embryos, the expression of both versican a and hyaluronan synthase 2 (has2) was highly upregulated, but the expression of bone morphogenetic protein 4 (bmp4) and T-box 2b (tbx2b) in the myocardium and of notch1b in the endocardium in the AVC did not change. Taken together, these results indicate that crip2 plays an important role in AV valve development by downregulating the expression of ECM components in the endocardial cushion.     

Essential functions of Alk3 during AV cushion morphogenesis in mouse embryonic hearts

Accumulated evidence has suggested that BMP pathways play critical roles during mammalian cardiogenesis and impairment of BMP signaling may contribute to human congenital heart diseases (CHDs), which are the leading cause of infant morbidity and mortality. Alk3 encodes a BMP specific type I receptor expressed in mouse embryonic hearts. To reveal functions of Alk3 during atrioventricular (AV) cushion morphogenesis and to overcome the early lethality of Alk3(-/-) embryos, we applied a Cre/loxp approach to specifically inactivate Alk3 in the endothelium/endocardium. Our studies showed that endocardial depletion of Alk3 severely impairs epithelium-mesenchymal-transformation (EMT) in the atrioventricular canal (AVC) region; the number of mesenchymal cells formed in Tie1-Cre;Alk3(loxp/loxp) embryos was reduced to only approximately 20% of the normal level from both in vivo section studies and in vitro explant assays. We showed, for the first time, that in addition to its functions on mesenchyme formation, Alk3 is also required for the normal growth/survival of AV cushion mesenchymal cells. Functions of Alk3 are accomplished through regulating expression/activation/subcellular localization of multiple downstream genes including Smads and cell-cycle regulators. Taken together, our study supports the notion that Alk3-mediated BMP signaling in AV endocardial/mesenchymal cells plays a central role during cushion morphogenesis.