Regulation of ascorbate-glutathione cycle by exogenous nitric oxide and hydrogen peroxide in soybean roots under arsenate stress.

The role of nitric oxide (NO) and hydrogen peroxide (H2O2) is well known for regulating plant abiotic stress responses. However, underlying mechanisms are still poorly understood. Therefore, the present study investigated the involvement of NO and H2O2 signalling in the regulation of arsenate toxicity (AsV) in soybean roots employing a pharmacological approach. Results show that AsV toxicity declined root length and biomass due to greater As accumulation in the cell wall and cellular organelles. Arsenate induced cell death due to enhanced levels of reactive oxygen species, lipid and protein oxidation and down-regulation in ascorbate-glutathione cycle and redox states of ascorbate and glutathione. These results correlate with lower endogenous level of NO. Interestingly, addition of L-NAME increased AsV toxicity. However, addition of SNP reverses effect of L-NAME, suggesting that endogenous NO has a role in mitigating AsV toxicity. Exogenous H2O2 also demonstrated capability of alleviating AsV stress, while NAC reversed the protective effect of H2O2. Furthermore, DPI application further increased AsV toxicity, suggesting that endogenous H2O2 is also implicated in mitigating AsV stress. SNP was not able to mitigate AsV toxicity in the presence of DPI, suggesting that H2O2 might have acted downstream of NO in accomplishing amelioration of AsV toxicity.

Full sunlight acclimation mechanisms in Riccia discolor thalli: Assessment at morphological, anatomical, and biochemical levels.

Light occupies a central position in regulating development of plants. Either little or excess of light could be harmful for plants. Since bryophytes are shade loving organisms, they must adapt to function in fluctuating light regimes. Therefore, the aim of this study was to investigate acclimatory responses of Riccia discolor thalli grown under full sunlight, and were compared with shade grown thalli (control). Length, width, and fresh mass of thallus were significantly lower (by 27, 41 and 37%, respectively) but endogenous nitric oxide content (by 81%) and nitric oxide synthase like activity (by 58%) were higher in full sunlight grown thalli than shade grown thalli. Number of rhizoids was greater in shade but length and width of rhizoids were higher (by 36 and 25%, respectively) in full sunlight grown thalli. The content of carotenoids was higher (by 34%) in full sunlight grown thalli. In full sunlight grown thalli, chloroplasts exhibited avoidance movement but in shade grown thalli they exhibited accumulation movement. Photosynthetic yields were higher in shade grown thalli. Among energy fluxes, ABS/RC did not vary but DI0/RC was higher (by 12%) in full sunlight grown thalli. Reactive oxygen species and damage were greater in full sunlight grown thalli despite enhanced levels of antioxidants i.e. superoxide dismutase (by 66%) and catalase (by 34%). Overall results suggest that full sunlight acclimation in Riccia discolor thalli occurred at various levels in which endogenous NO plays a positive role.

Wnt10b Gain-of-Function Improves Cardiac Repair by Arteriole Formation and Attenuation of Fibrosis.

RATIONALE: Myocardial infarction causes irreversible tissue damage, leading to heart failure. We recently discovered that canonical Wnt signaling and the Wnt10b ligand are strongly induced in mouse hearts after infarction. Wnt10b regulates cell fate in various organs, but its role in the heart is unknown. OBJECTIVE: To investigate the effect of Wnt10b gain-of-function on cardiac repair mechanisms and to assess its potential to improve ventricular function after injury. METHODS AND RESULTS: Histological and molecular analyses showed that Wnt10b is expressed in cardiomyocytes and localized in the intercalated discs of mouse and human hearts. After coronary artery ligation or cryoinjury in mice, Wnt10b is strongly and transiently induced in peri-infarct cardiomyocytes during granulation tissue formation. To determine the effect of Wnt10b on neovascularization and fibrosis, we generated a mouse line to increase endogenous Wnt10b levels in cardiomyocytes. We found that gain of Wnt10b function orchestrated a recovery phenotype characterized by robust neovascularization of the injury zone, less myofibroblasts, reduced scar size, and improved ventricular function compared with wild-type mice. Wnt10b stimulated expression of vascular endothelial growth factor receptor 2 in endothelial cells and angiopoietin-1 in vascular smooth muscle cells through nuclear factor-κB activation. These effects coordinated endothelial growth and smooth muscle cell recruitment, promoting robust formation of large, coronary-like blood vessels. CONCLUSION: Wnt10b gain-of-function coordinates arterial formation and attenuates fibrosis in cardiac tissue after injury. Because generation of mature blood vessels is necessary for efficient perfusion, our findings could lead to novel strategies to optimize the inherent repair capacity of the heart and prevent the onset of heart failure.

Gremlin 2 promotes differentiation of embryonic stem cells to atrial fate by activation of the JNK signaling pathway.

The bone morphogenetic protein antagonist Gremlin 2 (Grem2) is required for atrial differentiation and establishment of cardiac rhythm during embryonic development. A human Grem2 variant has been associated with familial atrial fibrillation, suggesting that abnormal Grem2 activity causes arrhythmias. However, it is not known how Grem2 integrates into signaling pathways to direct atrial cardiomyocyte differentiation. Here, we demonstrate that Grem2 expression is induced concurrently with the emergence of cardiovascular progenitor cells during differentiation of mouse embryonic stem cells (ESCs). Grem2 exposure enhances the cardiogenic potential of ESCs by 20-120-fold, preferentially inducing genes expressed in atrial myocytes such as Myl7, Nppa, and Sarcolipin. We show that Grem2 acts upstream to upregulate proatrial transcription factors CoupTFII and Hey1 and downregulate atrial fate repressors Irx4 and Hey2. The molecular phenotype of Grem2-induced atrial cardiomyocytes was further supported by induction of ion channels encoded by Kcnj3, Kcnj5, and Cacna1d genes and establishment of atrial-like action potentials shown by electrophysiological recordings. We show that promotion of atrial-like cardiomyocytes is specific to the Gremlin subfamily of BMP antagonists. Grem2 proatrial differentiation activity is conveyed by noncanonical BMP signaling through phosphorylation of JNK and can be reversed by specific JNK inhibitors, but not by dorsomorphin, an inhibitor of canonical BMP signaling. Taken together, our data provide novel mechanistic insights into atrial cardiomyocyte differentiation from pluripotent stem cells and will assist the development of future approaches to study and treat arrhythmias.

Continuous antagonism by Dkk1 counter activates canonical Wnt signaling and promotes cardiomyocyte differentiation of embryonic stem cells.

Embryonic stem (ES) cells give rise to mesodermal progenitors that differentiate to hematopoietic and cardiovascular cells. The wnt signaling pathway plays multiple roles in cardiovascular development through a network of intracellular effectors. To monitor global changes in wnt signaling during ES cell differentiation, we generated independent ES cell lines carrying the luciferase gene under promoters that uniquely respond to specific wnt pathway branches. Our results show that successive, mutually exclusive waves of noncanonical and canonical wnt signaling precede mesoderm differentiation. Blocking the initial noncanonical JNK/AP-1 signaling with SP60125 aborts cardiovascular differentiation and promotes hematopoiesis, whereas interference with the subsequent peak of canonical wnt signaling using Dkk1 has the opposite effect. Dkk1 blockade triggers counter mechanisms that lead to delayed and extended activation of canonical wnt signaling and mesoderm differentiation that appear to favor the cardiomyocytic lineage at the expense of hematopoietic cells. The cardiomyocytic yield can be further enhanced by overexpression of Wnt11 leading to approximately 95-fold enrichment in contracting cells. Our results suggest that the initial noncanonical wnt signaling is necessary for subsequent activation of canonical signaling and that the latter operates under a regulatory loop which responds to suppression with hyperactivation of compensatory mechanisms. This model provides new insights on wnt signaling during ES cell differentiation and points to a method to induce cardiomyocytic differentiation without precise timing of wnt signaling manipulation. Taking into account the heterogeneity of pluripotent cells, these findings might present an advantage to enhance the cardiogenic potential of stem cells.

Experimental myocardial infarction triggers canonical Wnt signaling and endothelial-to-mesenchymal transition.

Despite available therapies, myocardial infarction (MI) remains a leading cause of death worldwide. Better understanding of the molecular and cellular mechanisms that regulate cardiac repair should help to improve the clinical outcome of MI patients. Using the reporter mouse line TOPGAL, we show that canonical (ß-catenin-dependent) Wnt signaling is induced 4 days after experimental MI in subepicardial endothelial cells and perivascular smooth muscle actin (SMA)-positive (SMA(+)) cells. At 1 week after ischemic injury, a large number of canonical-Wnt-positive cells accumulated in the infarct area during granulation tissue formation. Coincidently with canonical Wnt activation, endothelial-to-mesenchymal transition (EndMT) was also triggered after MI. Using cell lineage tracing, we show that a significant portion of the canonical-Wnt-marked SMA(+) mesenchymal cells is derived from endothelial cells. Canonical Wnt signaling induces mesenchymal characteristics in cultured endothelial cells, suggesting a direct role in EndMT. In conclusion, our study demonstrates that canonical Wnt activation and EndMT are molecular and cellular responses to MI and that canonical Wnt signaling activity is a characteristic property of EndMT-derived mesenchymal cells that take part in cardiac tissue repair after MI. These findings could lead to new strategies to improve the course of cardiac repair by temporal and cell-type-specific manipulation of canonical Wnt signaling.

The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation.

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the "Functional Genomics in Embryonic Stem Cells" consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in "Expression Waves" and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells.

Disruption of the ifkA and ifkB genes results in altered cell adhesion, morphological defects and a propensity to form pre-stalk O cells during development of Dictyostelium.

IfkA and ifkB are two GCN2-like genes present in Dictyostelium. Disruption of either gene alone results in subtle developmental defects. However, disruption of ifkA and ifkB within the same strain results in severe morphological and patterning defects in the developing double null cells. The mutant cells aggregate in streams that give tightly clumped mounds. Fingers form from the mounds but remain attached to one another, especially at their bases. The fingers culminate to give fused and entangled structures lacking proper stalk but containing some spores. The morphological defects are consistent with an enhanced cell-cell and cell-substrate adhesiveness of the developing double null cells, which may result in inappropriate cell contacts and altered cell motility and sorting properties. In ifkA/ifkB nulls, cell type proportioning and patterning is altered in favor of ALC/pstO cell types. The bias toward the ALC/pstO cell types may be due, in part, to the nuclear localization of the transcription factor STATc in growing ifkA/ifkB null cells. STATc normally becomes localized to the nucleus during finger formation and only within the pre-stalk O zone. The precocious nuclear localization seen in the mutant cells may predispose the cells to a ALC/pstO cell fate. The findings indicate that IfkA and IfkB have redundant functions in Dictyostelium morphogenesis that involve maintaining proper cell-cell and cell-substrate adhesion and the equilibrium between different cell types for proper spatial patterning.

Ammonium transporter C of Dictyostelium discoideum is required for correct prestalk gene expression and for regulating the choice between slug migration and culmination.

Ammonium transporter C (AmtC) is one of three transporters in Dictyostelium that have been proposed to regulate entry and exit of ammonia in a cell type dependent manner and to mediate ammonia signaling. Previous work demonstrated that disruption of the amtC gene results in a slugger phenotype in which the cells remain as migrating slugs when they should form fruiting bodies. More detailed studies on the null strain revealed that differentiation of prestalk cell types was delayed and maintenance of prestalk cell gene expression was defective. There was little or no expression of ecmB, a marker for the initiation of culmination. Normal expression of CudA, a nuclear protein required for culmination, was absent in the anterior prestalk zone. The absence of CudA within the tip region was attributable to the lack of nuclear localization of the transcription factor STATa, despite expression of adenylyl cyclase A mRNA in the slug tips. Disruption of the histidine kinase gene dhkC in the amtC null strain restored STATa and CudA expression and the ability to culminate. The results suggest that the lack of nuclear translocation of STATa results from low cAMP due to a misregulated and overactive DhkC phosphorelay in the amtC null strain.