Bingruo Wu

Endocardial Cells Form the Coronary Arteries by Angiogenesis through Myocardial-Endocardial VEGF Signaling

The origins and developmental mechanisms of coronary arteries are incompletely understood. We show here by fate mapping, clonal analysis, and immunohistochemistry that endocardial cells generate the endothelium of coronary arteries. Dye tracking, live imaging, and tissue transplantation also revealed that ventricular endocardial cells are not terminally differentiated; instead, they are angiogenic and form coronary endothelial networks. Myocardial Vegf-a or endocardial Vegfr-2 deletion inhibited coronary angiogenesis and arterial formation by ventricular endocardial cells. In contrast, lineage and knockout studies showed that endocardial cells make a small contribution to the coronary veins, the formation of which is independent of myocardial-to-endocardial Vegf signaling. Thus, contrary to the current view of a common source for the coronary vessels, our findings indicate that the coronary arteries and veins have distinct origins and are formed by different mechanisms. This information may help develop better cell therapies for coronary artery disease.

Regulation of the Murine Nfatc1 Gene by NFATc2

NFAT proteins play a key role in the inducible expression of cytokine genes in T lymphocytes. NFATc1 and NFATc2 are the predominant NFAT family members in the peripheral immune system. NFATc2 is found abundantly in the cytoplasm of resting T cells, whereasNfatc1 expression is induced during T cell activation. To investigate Nfatc1 regulation, we characterized the structure of the murine Nfatc1 gene and its 5′-flanking region. A 290-bp sequence proximal to the transcription start site is highly conserved between mouse and human and possesses both basal and inducible promoter activities. Multiple binding sites for transcription factors were identified within this region, including a consensus NFAT-binding site. This promoter segment was cyclosporin A-sensitive, and mutation of the NFAT site abrogated inducible promoter activity and inhibited formation of an inducible DNA·protein complex containing NFATc2 in primary T cells. Overexpression of NFATc2 increased inducibleNfatc1 promoter activity, whereas this inducibility was attenuated in NFATc2−/− splenocytes. This study suggests that pre-existing NFATc2 contributes to the subsequent induction ofNfatc1 during T cell activation.

Nfatc1 Coordinates Valve Endocardial Cell Lineage Development Required for Heart Valve Formation

Rationale: Formation of heart valves requires early endocardial to mesenchymal transformation (EMT) to generate valve mesenchyme and subsequent endocardial cell proliferation to elongate valve leaflets. Nfatc1 (nuclear factor of activated T cells, cytoplasmic 1) is highly expressed in valve endocardial cells and is required for normal valve formation, but its role in the fate of valve endocardial cells during valve development is unknown. Objective: Our aim was to investigate the function of Nfatc1 in cell-fate decision making by valve endocardial cells during EMT and early valve elongation. Methods and Results: Nfatc1 transcription enhancer was used to generate a novel valve endocardial cell–specific Cre mouse line for fate-mapping analyses of valve endocardial cells. The results demonstrate that a subpopulation of valve endocardial cells marked by the Nfatc1 enhancer do not undergo EMT. Instead, these cells remain within the endocardium as a proliferative population to support valve leaflet extension. In contrast, loss of Nfatc1 function leads to enhanced EMT and decreased proliferation of valve endocardium and mesenchyme. The results of blastocyst complementation assays show that Nfatc1 inhibits EMT in a cell-autonomous manner. We further reveal by gene expression studies that Nfatc1 suppresses transcription of Snail1 and Snail2, the key transcriptional factors for initiation of EMT. Conclusions: These results show that Nfatc1 regulates the cell-fate decision making of valve endocardial cells during valve development and coordinates EMT and valve elongation by allocating endocardial cells to the 2 morphological events essential for valve development.

Characterization of Nfatc1 regulation identifies an enhancer required for gene expression that is specific to pro-valve endocardial cells in the developing heart

Nfatc1 is an endocardial transcription factor required for development of cardiac valves. Herein, we describe identification and characterization of a tissue-specific enhancer in the first intron of murine Nfatc1 that activates a heterogenic promoter and directs gene expression in a subpopulation of endocardial cells of the developing heart: the pro-valve endocardial cells. This enhancer activity begins on embryonic day (E) 8.5 in endocardial cells at the ventricular end of the atrioventricular canal, intensifies and extends from E9.5 to E11.5 in endocardium along the atrioventricular canal and outflow tract. By E12.5, the enhancer activity is accentuated in endocardial cells of forming valves. Sequential deletion analysis identified that a 250 bp DNA fragment at the 3′ end of the intron 1 is required for endocardial-specific activity. This region contains two short conserved sequences hosting a cluster of binding sites for transcription factors, including Nfat and Hox proteins. Electrophoresis mobility shift and chromatin immunoprecipitation assays demonstrated binding of Nfatc1 to the Nfat sites, and inactivation of Nfatc1 downregulated the enhancer activity in pro-valve endocardial cells. By contrast, mutation of the Hox site abolished its specificity, allowing gene expression in non pro-valve endocardium and extracardiac vasculature. Thus, autoregulation of Nfatc1 is required for maintaining high Nfatc1 expression in pro-valve endocardial cells, while suppression through the Hox site prevents its expression outside pro-valve endocardial cells during valve development. Our data demonstrate the first autonomous cell-specific enhancer for pro-valve endocardial cells and delineate a unique transcriptional mechanism that regulates endocardial Nfatc1 expression within developing cardiac valves.

Fog2 is critical for cardiac function and maintenance of coronary vasculature in the adult mouse heart

Aberrant transcriptional regulation contributes to the pathogenesis of both congenital and adult forms of heart disease. While the transcriptional regulator friend of Gata 2 (FOG2) is known to be essential for heart morphogenesis and coronary development, its tissue-specific function has not been previously investigated. Additionally, little is known about the role of FOG2 in the adult heart. Here we used spatiotemporally regulated inactivation of Fog2 to delineate its function in both the embryonic and adult mouse heart. Early cardiomyocyte- restricted loss of Fog2 recapitulated the cardiac and coronary defects of the Fog2 germline murine knockouts. Later cardiomyocyte-restricted loss of Fog2 (Fog2MC) did not result in defects in cardiac structure or coronary vessel formation. However, Fog2MC adult mice had severely depressed ventricular function and died at 8-14 weeks. Fog2MC adult hearts displayed a paucity of coronary vessels, associated with myocardial hypoxia, increased cardiomyocyte apoptosis, and cardiac fibrosis. Induced inactivation of Fog2 in the adult mouse heart resulted in similar phenotypes, as did ablation of the FOG2 interaction with the transcription factor GATA4. Loss of the FOG2 or FOG2-GATA4 interaction altered the expression of a panel of angiogenesis-related genes. Collectively, our data indicate that FOG2 regulates adult heart function and coronary angiogenesis.

Tbx20 acts upstream of Wnt signaling to regulate endocardial cushion formation and valve remodeling during mouse cardiogenesis

Cardiac valves are essential to direct forward blood flow through the cardiac chambers efficiently. Congenital valvular defects are prevalent among newborns and can cause an immediate threat to survival as well as long-term morbidity. Valve leaflet formation is a rigorously programmed process consisting of endocardial epithelial-mesenchymal transformation (EMT), mesenchymal cell proliferation, valve elongation and remodeling. Currently, little is known about the coordination of the diverse signals that regulate endocardial cushion development and valve elongation. Here, we report that the T-box transcription factor Tbx20 is expressed in the developing endocardial cushions and valves throughout heart development. Ablation of Tbx20 in endocardial cells causes severe valve elongation defects and impaired cardiac function in mice. Our study reveals that endocardial Tbx20 is crucial for valve endocardial cell proliferation and extracellular matrix development, but is not required for initiation of EMT. Elimination of Tbx20 also causes aberrant Wnt/β-catenin signaling in the endocardial cushions. In addition, Tbx20 regulates Lef1, a key transcriptional mediator for Wnt/β-catenin signaling, in this developmental process. Our study suggests a model in which Tbx20 regulates the Wnt pathway to direct endocardial cushion maturation and valve elongation, and provides new insights into the etiology of valve defects in humans.

Brg1 Governs a Positive Feedback Circuit in the Hair Follicle for Tissue Regeneration and Repair

Hair follicle stem cells (bulge cells) are essential for hair regeneration and early epidermal repair after wounding. Here we show that Brg1, a key enzyme in the chromatin-remodeling machinery, is dynamically expressed in bulge cells to control tissue regeneration and repair. In mice, sonic hedgehog (Shh) signals Gli to activate Brg1 in bulge cells to begin hair regeneration, whereas Brg1 recruits NF-κB to activate Shh in matrix cells to sustain hair growth. Such reciprocal Brg1-Shh interaction is essential for hair regeneration. Moreover, Brg1 is indispensable for maintaining the bulge cell reservoir. Without Brg1, bulge cells are depleted over time, partly through the ectopic expression of the cell-cycle inhibitor p27(Kip1). Also, bulge Brg1 is activated by skin injury to facilitate early epidermal repair. Our studies demonstrate a molecular circuit that integrates chromatin remodeling (Brg1), transcriptional regulation (NF-κB, Gli), and intercellular signaling (Shh) to control bulge stem cells during tissue regeneration.

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.

DNA Methylation is Developmentally Regulated for Genes Essential for Cardiogenesis

Background DNA methylation is a major epigenetic mechanism altering gene expression in development and disease. However, its role in the regulation of gene expression during heart development is incompletely understood. The aim of this study is to reveal DNA methylation in mouse embryonic hearts and its role in regulating gene expression during heart development. Methods and Results We performed the genome‐wide DNA methylation profiling of mouse embryonic hearts using methyl‐sensitive, tiny fragment enrichment/massively parallel sequencing to determine methylation levels at ACGT sites. The results showed that while global methylation of 1.64 million ACGT sites in developing hearts remains stable between embryonic day (E) 11.5 and E14.5, a small fraction (2901) of them exhibit differential methylation. Gene Ontology analysis revealed that these sites are enriched at genes involved in heart development. Quantitative real‐time PCR analysis of 350 genes with differential DNA methylation showed that the expression of 181 genes is developmentally regulated, and 79 genes have correlative changes between methylation and expression, including hyaluronan synthase 2 (Has2). Required for heart valve formation, Has2 expression in the developing heart valves is downregulated at E14.5, accompanied with increased DNA methylation in its enhancer. Genetic knockout further showed that the downregulation of Has2 expression is dependent on DNA methyltransferase 3b, which is co‐expressed with Has2 in the forming heart valve region, indicating that the DNA methylation change may contribute to the Has2 enhancers regulating function. Conclusions DNA methylation is developmentally regulated for genes essential to heart development, and abnormal DNA methylation may contribute to congenital heart disease.

Inducible Cardiomyocyte-Specific Gene Disruption Directed by the Rat Tnnt2 Promoter in the Mouse

We developed a conditional and inducible gene knockout methodology that allows effective gene deletion in mouse cardiomyocytes. This transgenic mouse line was generated by coinjection of two transgenes, a “reverse” tetracycline‐controlled transactivator (rtTA) directed by a rat cardiac troponin T (Tnnt2) promoter and a Cre recombinase driven by a tetracycline‐responsive promoter (TetO). Here, Tnnt2‐rtTA activated TetO‐Cre expression takes place in cardiomyocytes following doxycycline treatment. Using two different mouse Cre reporter lines, we demonstrated that expression of Cre recombinase was specifically and robustly induced in the cardiomyocytes of embryonic or adult hearts following doxycycline induction, thus, allowing cardiomyocyte‐specific gene disruption and lineage tracing. We also showed that rtTA expression and doxycycline treatment did not compromise cardiac function. These features make the Tnnt2‐rtTA;TetO‐Cre transgenic line a valuable genetic tool for analysis of spatiotemporal gene function and cardiomyocyte lineage tracing during developmental and postnatal periods. genesis 48:63–72, 2010.