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Atrioventricular valve development requires endothelial-to-mesenchymal transition (EndMT) that induces cushion endocardial cells to give rise to mesenchymal cells crucial to valve formation. In the adult endothelium, deletion of the docking protein FRS2α induces EndMT by activating TGFβ signaling in a miRNA let-7-dependent manner. To study the role of endothelial FRS2α during embryonic development, we generated mice with an inducible endothelial-specific deletion of Frs2α (FRS2α iECKO ). Analysis of the FRS2α iECKO embryos uncovered a combination of impaired EndMT in AV cushions and defective maturation of AV valves leading to development of thickened, abnormal valves when Frs2α was deleted early (E7.5) in development. At the same time, no AV valve developmental abnormalities were observed after late (E10.5) deletion. These observations identify FRS2α as a pivotal controller of cell fate transition during both EndMT and post-EndMT valvulogenesis., Competing Interests: Declaration of competing interest None., (Copyright © 2019. Published by Elsevier Inc.)

Heart formation involves a complex series of tissue rearrangements, during which regions of the developing organ expand, bend, converge, and protrude in order to create the specific shapes of important cardiac components. Much of this morphogenesis takes place while cardiac function is underway, with blood flowing through the rapidly contracting chambers. Fluid forces are therefore likely to influence the regulation of cardiac morphogenesis, but it is not yet clear how these biomechanical cues direct specific cellular behaviors. In recent years, the optical accessibility and genetic amenability of zebrafish embryos have facilitated unique opportunities to integrate the analysis of flow parameters with the molecular and cellular dynamics underlying cardiogenesis. Consequently, we are making progress toward a comprehensive view of the biomechanical regulation of cardiac chamber emergence, atrioventricular canal differentiation, and ventricular trabeculation. In this review, we highlight a series of studies in zebrafish that have provided new insight into how cardiac function can shape cardiac morphology, with a particular focus on how hemodynamics can impact cardiac cell behavior. Over the long-term, this knowledge will undoubtedly guide our consideration of the potential causes of congenital heart disease., (© 2019 Elsevier Inc. All rights reserved.)

Background: The origin of the intercalated cushions that develop into the anterior cusp of the pulmonary valve (PV) and the noncoronary cusp of the aortic valve (AV) is not well understood., Results: Cre transgenes in combination with the Rosa TdTomato-EGFP reporter were used to generate three-dimensional lineage mapping of AV and PV cusps during intercalated cushion development. Tie2-Cre;EGFP was used to mark endothelial-derived mesenchymal cells, Wnt1-Cre;EGFP for cardiac neural crest and cardiac Troponin T (Tnnt2)Cre;EGFP, for myocardial lineage. The highest percentage of intercalated cushion cells at embryonic day (E) 12.5 was Tnnt2-Cre; EGFP positive; 68.0% for the PV and 50.0% AV. Neither Tnnt2 mRNA nor Tnnt2-Cre protein was expressed in the intercalated cushions; and the Tnnt2-Cre lineage intercalated cushion cells were also positive for the mesenchymal markers Sox9 and versican. Tnnt2-Cre lineage was present within the forming intercalated cushions from E11.5 and was present in the intercalated cushion derived PV and AV cusps and localized to the fibrosa layer at postnatal day 0., Conclusions: Intercalated cushions of the developing outflow tract are populated with Tnnt2-Cre derived cells, a Cre reporter previously used for tracing and excision of myocardial cells and not previously associated with mesenchymal cells. Developmental Dynamics 247:1005-1017, 2018. © 2018 Wiley Periodicals, Inc., (© 2018 Wiley Periodicals, Inc.)

Nephronectin (NPNT), a highly conserved extracellular matrix protein, plays an important role in regulating cell adhesion, differentiation, spreading, and survival. NPNT protein belongs to the epidermal growth factor (EGF)-like superfamily and exhibits several common structural determinants; including EGF-like repeat domains, MAM domain (Meprin, A5 Protein, and Receptor Protein-Tyrosine Phosphatase µ), RGD motif (Arg-Gly-Asp) and a coiled-coil domain. It regulates integrins-mediated signaling pathways via the interaction of its RGD motif with integrin α8β1. Recent studies revealed that NPNT is involved in kidney development, renal injury repair, atrioventricular canal differentiation, pulmonary function, and muscle cell niche maintenance. Moreover, NPNT regulates osteoblast differentiation and mineralization, as well as osteogenic angiogenesis. Altered expression of NPNT has been linked with the progression of certain types of cancers, such as spontaneous breast tumor metastasis and malignant melanoma. Interestingly, NPNT gene expression can be regulated by a range of external factors such as tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), oncostatin M (OSM), bone morphogenic protein 2 (BMP2), Wnt3a, Vitamin D 3 , and microRNA-378 (miR378). Further understanding the cellular and molecular mechanisms by which NPNT regulates tissue homeostasis in an organ-specific manner is critical in exploring NPNT as a therapeutic target for tissue regeneration and tissue engineering., (© 2017 Wiley Periodicals, Inc.)

Myocardial contractility and blood flow provide essential mechanical cues for the morphogenesis of the heart. In general, endothelial cells change their migratory behavior in response to shear stress patterns, according to flow directionality. Here, we assessed the impact of shear stress patterns and flow directionality on the behavior of endocardial cells, the specialized endothelial cells of the heart. At the early stages of zebrafish heart valve formation, we show that endocardial cells are converging to the valve-forming area and that this behavior depends upon mechanical forces. Quantitative live imaging and mathematical modeling allow us to correlate this tissue convergence with the underlying flow forces. We predict that tissue convergence is associated with the direction of the mean wall shear stress and of the gradient of harmonic phase-averaged shear stresses, which surprisingly do not match the overall direction of the flow. This contrasts with the usual role of flow directionality in vascular development and suggests that the full spatial and temporal complexity of the wall shear stress should be taken into account when studying endothelial cell responses to flow in vivo ., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKO Endo ) by crossing conditionally-targeted Bmp2 flox/flox mice with a Cre-driver line, Nfatc1 Cre , wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKO Endo mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5-11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKO Endo mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKO Endo mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKO Endo in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKO Endo AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKO Endo AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Heart valve development proceeds through coordinated steps by which endocardial cushions (ECs) form thin, elongated and stratified valves. Wnt signaling and its canonical effector β-catenin are proposed to contribute to endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define specific roles of the canonical Wnt pathway at different stages of valve formation. We developed a transgenic mouse system that provides spatiotemporal inhibition of Wnt/β-catenin signaling by chemically inducible overexpression of Dkk1. Unexpectedly, this approach indicates canonical Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Furthermore, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt/β-catenin signaling is activated in cushion mesenchymal cells where it supports FGF-driven expansion of ECs and then AVC valve extracellular matrix patterning. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that accumulating Axin2 in maturing valves represents negative feedback that restrains tissue overgrowth rather than simply reporting Wnt activity. Disruption of these Wnt/β-catenin signaling roles that enable developmental transitions during valvulogenesis could account for common congenital valve defects., (© 2016. Published by The Company of Biologists Ltd.)

Endothelial to mesenchymal transition (EMT) that occurs during cardiac outflow tract (OFT) development is critical for formation of the semilunar valves. Fibulin-1 (Fbln1) is an extracellular matrix protein that is present at several sites of EMT, including the OFT (i.e., E9.5-10.5). The aim of this study was to determine the role of Fbln1 in EMT during the earliest events of OFT development. Examination of proximal OFT cushions in Fbln1 null embryos detected hypercellularity at both E9.5 (93% increase; p = 0.002) and E10.5 (43% increase; p = 0.01) as compared to wild type, suggesting that Fbln1 normally suppresses OFT endocardial cushion EMT. This was supported by studies of proximal OFT cushion explants, which showed that explants from Fbln1 null embryos displayed a 58% increase in cells migrating from the explants as compared to wild type (p = 0.005). We next evaluated the effects of Fbln1 deficiency on the expression of factors that regulate proximal OFT EMT. At E9.5, Fbln1 null proximal OFT endocardium and EMT-derived mesenchyme showed increased TGFβ2 (58% increase; p = 0.01) and increased Snail1-positive nuclei (27% increase; p = 0.0003). Histological examination of OFT cushions in Fbln1 null embryos (E9.5) also detected cells present in the cushion that were determined to be erythrocytes based on round morphology, autofluorescence, and positive staining for hemoglobin. Erythrocytes were also detected in Fbln1 null OFT cushions at E10.5. Together, the findings indicate that Fbln1 normally suppresses proximal OFT EMT preventing proximal cushion hypercellularity and blood cell accumulation., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Background: The basic helix-loop-helix transcription factor Twist1 has well-documented roles in progenitor populations of the developing embryo, including endocardial cushions (ECC) and limb buds, and also in cancer. Whether Twist1 regulates the same transcriptional targets in different tissue types is largely unknown., Results: The tissue-specificity of Twist1 genomic occupancy was examined in mouse ECCs, limb buds, and peripheral nerve sheath tumor (PNST) cells using chromatin immunoprecipitation followed by sequencing (Chip-seq) analysis. Consistent with known Twist1 functions during development and in cancer cells, Twist1-DNA binding regions associated with genes related to cell migration and adhesion were detected in all three tissues. However, the vast majority of Twist1 binding regions were specific to individual tissue types. Thus, while Twist1 has similar functions in ECCs, limb buds, and PNST cells, the specific genomic sequences occupied by Twist1 were different depending on cellular context. Subgroups of shared genes, also predominantly related to cell adhesion and migration, were identified in pairwise comparisons of ECC, limb buds and PNST cells. Twist1 genomic occupancy was detected for six binding regions in all tissue types, and Twist1-binding sequences associated with Chst11, Litaf, Ror2, and Spata5 also bound the potential Twist1 cofactor RREB1. Pathway analysis of the genes associated with Twist1 binding suggests that Twist1 may regulate genes associated with the Wnt signaling pathway in ECCs and limb buds., Conclusions: Together, these data indicate that Twist1 interacts with genes that regulate adhesion and migration in different tissues, potentially through distinct sets of target genes. In addition, there is a small subset of genes occupied by Twist1 in all three tissues that may represent a core group of Twist1 target genes in multiple cell types.

Cardiac malformations due to aberrant development of the atrioventricular (AV) valves are among the most common forms of congenital heart diseases. Normally, heart valve mesenchyme is formed from an endothelial to mesenchymal transition (EMT) of endothelial cells of the endocardial cushions. Yes-associated protein 1 (YAP1) has been reported to regulate EMT in vitro, in addition to its known role as a major regulator of organ size and cell proliferation in vertebrates, leading us to hypothesize that YAP1 is required for heart valve development. We tested this hypothesis by conditional inactivation of YAP1 in endothelial cells and their derivatives. This resulted in markedly hypocellular endocardial cushions due to impaired formation of heart valve mesenchyme by EMT and to reduced endocardial cell proliferation. In endothelial cells, TGFβ induces nuclear localization of Smad2/3/4 complex, which activates expression of Snail, Twist1, and Slug, key transcription factors required for EMT. YAP1 interacts with this complex, and loss of YAP1 disrupts TGFβ-induced up-regulation of Snail, Twist1, and Slug. Together, our results identify a role of YAP1 in regulating EMT through modulation of TGFβ-Smad signaling and through proliferative activity during cardiac cushion development., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aims: Anomalies of the arterial valves, principally bicuspid aortic valve (BAV), are the most common congenital anomalies. The cellular mechanisms that underlie arterial valve development are poorly understood. While it is known that the valve leaflets derive from the outflow cushions, which are populated by cells derived from the endothelium and neural crest cells (NCCs), the mechanism by which these cushions are sculpted to form the leaflets of the arterial valves remains unresolved. We set out to investigate how NCCs participate in arterial valve formation, reasoning that disrupting NCC within the developing outflow cushions would result in arterial valve anomalies, in the process elucidating the normal mechanism of arterial valve leaflet formation., Methods and Results: By disrupting Rho kinase signalling specifically in NCC using transgenic mice and primary cultures, we show that NCC condensation within the cardiac jelly is required for correct positioning of the outflow cushions. Moreover, we show that this process is essential for normal patterning of the arterial valve leaflets with disruption leading to a spectrum of valve leaflet patterning anomalies, abnormal positioning of the orifices of the coronary arteries, and abnormalities of the arterial wall., Conclusion: NCCs are required at earlier stages of arterial valve development than previously recognized, playing essential roles in positioning the cushions, and patterning the valve leaflets. Abnormalities in the process of NCC condensation at early stages of outflow cushion formation may provide a common mechanism underlying BAV, and also explain the link with arterial wall anomalies and outflow malalignment defects.

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 β (TGFβ) 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 TGFβ-dependent endocardial cell invasion., Results: In this study, we demonstrate that the effect of MBNL1 knockdown on invasion remains dependent on TGFβ3 after it is no longer required to induce basal levels of invasion. TGFβ3, but not TGFβ2, levels are elevated in medium conditioned by MBNL1-depleted AVC explants. TGFβ3 is elevated even when the myocardium is removed, indicating that MBNL1 modulates autocrine TGFβ3 production in the endocardium. More TGFβ3-positive cells are observed in the endocardial monolayer following MBNL1 knockdown. Addition of exogenous TGFβ3 to AVC explants recapitulates the effects of MBNL1 knockdown. Time course experiments demonstrate that knockdown of MBNL1 induces precocious TGFβ3 secretion, and early exposure to excess TGFβ3 induces precocious invasion. MBNL1 expression precedes TGFβ3 in the AVC endocardium, consistent with a role in preventing precocious autocrine TGFβ3 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. TGFβ 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 TGFβ3 production.

Atrioventricular valve development commences with an EMT event whereby endocardial cells transform into mesenchyme. The molecular events that induce this phenotypic change are well understood and include many growth factors, signaling components, and transcription factors. Besides their clear importance in valve development, the role of these transformed mesenchyme and the function they serve in the developing prevalve leaflets is less understood. Indeed, we know that these cells migrate, but how and why do they migrate? We also know that they undergo a transition to a mature, committed cell, largely defined as an interstitial fibroblast due to their ability to secrete various matrix components including collagen type I. However, we have yet to uncover mechanisms by which the matrix is synthesized, how it is secreted, and how it is organized. As valve disease is largely characterized by altered cell number, cell activation, and matrix disorganization, answering questions of how the valves are built will likely provide us with information of real clinical relevance. Although expression profiling and descriptive or correlative analyses are insightful, to advance the field, we must now move past the simplicity of these assays and ask fundamental, mechanistic based questions aimed at understanding how valves are "built". Herein we review current understandings of atrioventricular valve development and present what is known and what isn't known. In most cases, basic, biological questions and hypotheses that were presented decades ago on valve development still are yet to be answered but likely hold keys to uncovering new discoveries with relevance to both embryonic development and the developmental basis of adult heart valve diseases. Thus, the goal of this review is to remind us of these questions and provide new perspectives on an old theme of valve development., (Published by Elsevier B.V.)

Semilunar valve malformations are common human congenital heart defects. Bicuspid aortic valves occur in 2-3% of the population, and pulmonic valve stenosis constitutes 10% of all congenital heart disease in adults (Brickner et al., 2000) [1]. Semilunar valve defects cause valve regurgitation, stenosis, or calcification, leading to endocarditis or congestive heart failure. These complications often require prolonged medical treatment or surgical intervention. Despite the medical importance of valve disease, the regulatory pathways governing semilunar valve development are not entirely clear. In this report we investigated the spatiotemporal role of calcineurin/Nfatc1 signaling in semilunar valve development. We generated conditional knockout mice with calcineurin gene disrupted in various tissues during semilunar valve development. Our studies showed that calcineurin/Nfatc1 pathway signals in the secondary heart field (SHF) but not in the outflow tract myocardium or neural crest cells to regulate semilunar valve morphogenesis. Without SHF calcineurin/Nfatc1 signaling, the conal endocardial cushions-the site of prospective semilunar valve formation--first develop and then regress due to apoptosis, resulting in a striking phenotype with complete absence of the aortic and pulmonic valves, severe valve regurgitation, and perinatal lethality. This role of calcineurin/Nfatc1 signaling in the SHF is different from the requirement of calcineurin/Nfatc1 in the endocardium for semilunar valve formation (Chang et al., 2004) [2], indicating that calcineurin/Nfatc1 signals in multiple tissues to organize semilunar valve development. Also, our studies suggest distinct mechanisms of calcineurin/Nfat signaling for semilunar and atrioventricular valve morphogenesis. Therefore, we demonstrate a novel developmental mechanism in which calcineurin signals through Nfatc1 in the secondary heart field to promote semilunar valve morphogenesis, revealing a new supportive role of the secondary heart field for semilunar valve formation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

An early event in heart valve formation is the epithelial-mesenchymal transformation (EMT) of a subpopulation of endothelial cells in specific regions of the heart tube, the endocardial cushions. The Type III TGFβ receptor (TGFβR3) is required for TGFβ2- or BMP-2-stimulated EMT in atrioventricular endocardial cushion (AVC) explants in vitro but the mediators downstream of TGFβR3 are not well described. Using AVC and ventricular explants as an in vitro assay, we found an absolute requirement for specific TGFβR3 cytoplasmic residues, GAIP-interacting protein, C terminus (GIPC), and specific Activin Receptor-Like Kinases (ALK)s for TGFβR3-mediated EMT when stimulated by TGFβ2 or BMP-2. The introduction of TGFβR3 into nontransforming ventricular endocardial cells, followed by the addition of either TGFβ2 or BMP-2, results in EMT. TGFβR3 lacking the entire cytoplasmic domain, or only the 3C-terminal amino acids that are required to bind GIPC, fails to support EMT in response to TGFβ2 or BMP-2. Overexpression of GIPC in AVC endocardial cells enhanced EMT while siRNA-mediated silencing of GIPC in ventricular cells overexpressing TGFβR3 significantly inhibited EMT. Targeting of specific ALKs by siRNA revealed that TGFβR3-mediated EMT requires ALK2 and ALK3, in addition to ALK5, but not ALK4 or ALK6. Taken together, these data identify GIPC, ALK2, ALK3, and ALK5 as signaling components required for TGFβR3-mediated endothelial cell EMT., (Copyright © 2011. Published by Elsevier Inc.)

Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development.

Cardiac valves originate from endocardial cushions (EC) formed by endothelial-to-mesenchymal transformation (EMT) during embryogenesis. The zinc-finger transcription factor Snai1 has previously been reported to be important for EMT during organogenesis, yet its role in early valve development has not been directly examined. In this study we show that Snai1 is highly expressed in endothelial, and newly transformed mesenchyme cells during EC development. Mice with targeted snai1 knockdown display hypocellular ECs at E10.5 associated with decreased expression of mesenchyme cell markers and downregulation of the matrix metalloproteinase (mmp) family member, mmp15. Snai1 overexpression studies in atrioventricular canal collagen I gel explants indicate that Snai1 is sufficient to promote mmp15 expression, cell transformation, and mesenchymal cell migration and invasion. However, treatment with the catalytically active form of MMP15 promotes cell motility, and not transformation. Further, we show that Snai1-mediated cell migration requires MMP activity, and caMMP15 treatment rescues attenuated migration defects observed in murine ECs following snai1 knockdown. Together, findings from this study reveal previously unappreciated mechanisms of Snai1 for the direct regulation of MMPs during EC development., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Rationale: Since their discovery almost 20 years ago, microRNAs have been shown to perform essential roles during tissue development and disease. Although roles for microRNAs in the myocardium during embryo development and cardiac disease have been demonstrated, very little is know about their role in the endocardium or during cardiac valve formation., Objective: To study the role of microRNAs in cardiac valve formation., Methods and Results: We show that zebrafish dicer mutant embryos, lacking mature miRNAs, form excessive endocardial cushions. By screening miRNAs expressed in the heart, we found that miR-23 is both necessary and sufficient for restricting the number of endocardial cells that differentiate into endocardial cushion cells. In addition, in mouse endothelial cells, miR-23 inhibited a transforming growth factor-β-induced endothelial-to-mesenchymal transition. By in silico screening of expression data with predicted miR-23 target sites combined with in vivo testing, we identified hyaluronic acid synthase 2 (Has2), Icat, and Tmem2 as novel direct targets of miR-23. Finally, we demonstrate that the upregulation of Has2, an extracellular remodeling enzyme required for endocardial cushion and valve formation, is responsible for the excessive endocardial cushion cell differentiation in dicer mutants., Conclusions: MiR-23 in the embryonic heart is required to restrict endocardial cushion formation by inhibiting Has2 expression and extracellular hyaluronic acid production.

The heart is the most common site of congenital defects, and valvuloseptal defects are the most common of the cardiac anomalies seen in the newborn. The process of endothelial-to-mesenchymal transition (EndMT) in the cardiac cushions is a required step during early valve development, and Notch signaling is required for this process. Here we show that Notch activation induces the transcription of both subunits of the soluble guanylyl cyclase (sGC) heterodimer, GUCY1A3 and GUCY1B3, which form the nitric oxide receptor. In parallel, Notch also promotes nitric oxide (NO) production by inducing Activin A, thereby activating a PI3-kinase/Akt pathway to phosphorylate eNOS. We thus show that the activation of sGC by NO through a Notch-dependent autocrine loop is necessary to drive early EndMT in the developing atrioventricular canal (AVC)., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Atrioventricular (AV) cushions are the precursors of AV septum and valves. In this study, we examined roles of Smad4 during AV cushion development using a conditional gene inactivation approach. We found that endothelial/endocardial inactivation of Smad4 led to the hypocellular AV cushion defect and that both reduced cell proliferation and increased apoptosis contributed to the defect. Expression of multiple genes critical for cushion development was down-regulated in mutant embryos. In collagen gel assays, the number of mesenchymal cells formed is significantly reduced in mutant AV explants compared to that in control explants, suggesting that the reduction of cushion mesenchyme formation in mutants is unlikely secondary to their gross vasculature abnormalities. Using a previously developed immortal endocardial cell line, we showed that Smad4 is required for BMP signaling- stimulated upregulation of Tbx20 and Gata4. Therefore, our data collectively support the cell-autonomous requirement of endocardial Smad4 in regulating AV cushion development., (© 2010 Wiley-Liss, Inc.)

Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase which has been shown to regulate cell migration upon binding its ligand, collagen. Expression studies determined that DDR2 mRNA and protein are present in the atrioventricular canal during epithelial-mesenchymal transformation (EMT) and the receptor is expressed in both activated endothelial and migrating mesenchymal cells in vivo.

Congenital heart defects occur at a rate of 5% and are the most prevalent birth defects. A better understanding of the complex signaling networks regulating heart development is necessary to improve repair strategies for congenital heart defects. The mitogen-activated protein 3 kinase (MEKK3) is important to early embryogenesis, but developmental processes affected by MEKK3 during heart morphogenesis have not been fully examined. We identify MEKK3 as a critical signaling molecule during endocardial cushion development. We report the detection of MEKK3 transcripts to embryonic hearts before, during, and after cardiac cushion cells have executed epithelial-to-mesenchymal transition (EMT). MEKK3 is observed to endocardial cells of the cardiac cushions with a diminishing gradient of expression into the cushions. These observations suggest that MEKK3 may function during production of cushion mesenchyme as required for valvular development and septation of the heart. We used a kinase inactive form of MEKK3 (MEKK3(KI)) in an in vitro assay that recapitulates in vivo EMT and show that MEKK3(KI) attenuates mesenchyme formation. Conversely, constitutively active MEKK3 (ca-MEKK3) triggers mesenchyme production in ventricular endocardium, a tissue that does not normally undergo EMT. MEKK3-driven mesenchyme production is further substantiated by increased expression of EMT-relevant genes, including TGFbeta(2), Has2, and periostin. Furthermore, we show that MEKK3 stimulates EMT via a TGFbeta(2)-dependent mechanism. Thus, the activity of MEKK3 is sufficient for developmental EMT in the heart. This knowledge provides a basis to understand how MEKK3 integrates signaling cascades activating endocardial cushion EMT.

The endocardial cushions play a critical role in septation of the four-chambered mammalian heart and in the formation of the valve leaflets that control blood flow through the heart. Within the outflow tract (OFT), both cardiac neural crest and endocardial-derived mesenchymal cells contribute to the endocardial cushions. Bone morphogenetic protein 4 (BMP4) is required for endocardial cushion development and for normal septation of the OFT. In the present study, we show that anterior heart field (AHF)-derived myocardium is an essential source of BMP4 required for normal endocardial cushion expansion and remodeling. Loss of BMP4 from the AHF in mice results in an insufficient number of cells in the developing OFT endocardial cushions, defective cushion remodeling, ventricular septal defects, persistent truncus arteriosus, and abnormal semilunar valve formation., (Copyright (c) 2008 Wiley-Liss, Inc.)

Trophic factors secreted both from the endocardium and epicardium regulate appropriate growth of the myocardium during cardiac development. Epicardially-derived cells play also a key role in development of the coronary vasculature. This process involves transformation of epithelial (epicardial) cells to mesenchymal cells (EMT). Similarly, a subset of endocardial cells undergoes EMT to form the mesenchyme of endocardial cushions, which function as primordia for developing valves and septa. While it has been suggested that transforming growth factor-betas (Tgf-beta) play an important role in induction of EMT in the avian epi- and endocardium, the function of Tgf-betas in corresponding mammalian tissues is still poorly understood. In this study, we have ablated the Tgf-beta type I receptor Alk5 in endo-, myo- and epicardial lineages using the Tie2-Cre, Nkx2.5-Cre, and Gata5-Cre driver lines, respectively. We show that while Alk5-mediated signaling does not play a major role in the myocardium during mouse cardiac development, it is critically important in the endocardium for induction of EMT both in vitro and in vivo. Moreover, loss of epicardial Alk5-mediated signaling leads to disruption of cell-cell interactions between the epicardium and myocardium resulting in a thinned myocardium. Furthermore, epicardial cells lacking Alk5 fail to undergo Tgf-beta-induced EMT in vitro. Late term mutant embryos lacking epicardial Alk5 display defective formation of a smooth muscle cell layer around coronary arteries, and aberrant formation of capillary vessels in the myocardium suggesting that Alk5 is controlling vascular homeostasis during cardiogenesis. To conclude, Tgf-beta signaling via Alk5 is not required in myocardial cells during mammalian cardiac development, but plays an irreplaceable cell-autonomous role regulating cellular communication, differentiation and proliferation in endocardial and epicardial cells.

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., Results: While both Msx1-/- and Msx2-/- single homozygous mutant mice exhibited normal valve formation, we observed hypoplastic AV cushions and malformed AV valves in Msx1-/-; Msx2-/- mutants, indicating redundant functions of Msx1 and Msx2 during AV valve morphogenesis. In Msx1/2 null mutant AV cushions, we found decreased Bmp2/4 and Notch1 signaling as well as reduced expression of Has2, NFATc1 and Notch1, demonstrating impaired endocardial activation and EMT. Moreover, perturbed expression of chamber-specific genes Anf, Tbx2, Hand1 and Hand2 reveals mispatterning of the Msx1/2 double mutant myocardium and suggests functions of Msx1 and Msx2 in regulating myocardial signals required for remodelling AV valves and maintaining an undifferentiated state of the AV myocardium., Conclusion: Our findings demonstrate redundant roles of Msx1 and Msx2 in regulating signals required for development of the AV myocardium and formation of the AV valves.

Snail family proteins are key regulators of epithelial-mesenchymal transition, but their role in endothelial-to-mesenchymal transition (EMT) is less well studied. We show that Slug, a Snail family member, is expressed by a subset of endothelial cells as well as mesenchymal cells of the atrioventricular canal and outflow tract during cardiac cushion morphogenesis. Slug deficiency results in impaired cellularization of the cardiac cushion at embryonic day (E)-9.5 but is compensated by increased Snail expression at E10.5, which restores cardiac cushion EMT. We further demonstrate that Slug, but not Snail, is directly up-regulated by Notch in endothelial cells and that Slug expression is required for Notch-mediated repression of the vascular endothelial cadherin promoter and for promoting migration of transformed endothelial cells. In contrast, transforming growth factor beta (TGF-beta) induces Snail but not Slug. Interestingly, activation of Notch in the context of TGF-beta stimulation results in synergistic up-regulation of Snail in endothelial cells. Collectively, our data suggest that combined expression of Slug and Snail is required for EMT in cardiac cushion morphogenesis.

Atrioventricular (AV) endocardium transforms into the cushion mesenchyme, the primordia of the valves and membranous septa, through epithelial-mesenchymal transformation (EMT). While bone morphogenetic protein (BMP)-2 is known to be critical for AV EMT, the role of BMP-2 in post-EMT AV valvulogenesis remains to be elucidated. To find BMP signaling loops, we first localized Type I BMP receptors (BMPRs), BMPR-1A (ALK3), -1B (ALK6) and ALK2 in AV cushion mesenchyme in stage-24 chick embryos. Based on the BMP receptor expression pattern, we examined the functional roles of BMP-2 and BMP signaling in post-EMT valvulogenesis by using stage-24 AV cushion mesenchymal cell aggregates cultured on 3D-collagen gels. Exogenous BMP-2 or constitutively active (ca) BMPR-1B (ALK6)-virus treatments induced migration of the mesenchymal cells into the collagen gels, whereas noggin, an antagonist of BMPs, or dominant-negative (dn) BMPR-1 B (ALK6)-virus treatments reduced cell migration from the mesenchymal cell aggregates. Exogenous BMP-2 or caBMPR-1B (ALK6) treatments significantly promoted expression of an extracellular matrix (ECM) protein, periostin, a known valvulogenic matrix maturation mediator, at both mRNA and protein levels, whereas periostin expression was repressed by adding noggin or dnBMPR-1B (ALK6)-virus to the culture. Moreover, transcripts of Twist and Id1, which have been implicated in cell migration in embryogenesis and activation of the periostin promoter, were induced by BMP-2 but repressed by noggin in cushion mesenchymal cell cultures. These data provide evidence that BMP-2 and BMP signaling induce biological processes involved in early AV valvulogenesis, i.e. mesenchymal cell migration and expression of periostin, indicating critical roles for BMP signaling in post-EMT AV cushion tissue maturation and differentiation.