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Diego Franco - One of the best experts on this subject based on the ideXlab platform.
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atrial spceific Pitx2 insufficiencyincreases the frequency of calcium sparks waves and after depolarizations in mouse atrial myocytes
Biophysical Journal, 2017Co-Authors: C Tarifa, Alexander Vallmitjana, Raul Benitez, Adela Herraizmartinez, Selma A Serra, Diego Franco, Leif HovemadsenAbstract:The Transcription Factor Pitx2 has been proposed as a molecular link between single nucleotide polymorphisms on chromosome 4q25 and increased risk of atrial fibrillation in carriers of the risk variant. Since atrial fibrillation has been associated with calcium handling disturbances in isolated atrial myocytes, we here tested the hypothesis that Pitx2 insufficiency alters the calcium homeostasis in atrial myocytes.To test this hypothesis, we used right atrial myocytes from a transgenic mouse model with inducible atrial specific Pitx2 deletion. Spontaneous calcium release was detected with confocal calcium imaging and resulting ion currents or membrane depolarizations were measured with patch-clamp technique in myocytes from wild-type (Pitx2+/+) and heterozygous Pitx2+/- mice.Calcium imaging revealed that the frequency of calcium sparks (2.1±0.7 vs 0.2±0.1 events/cell/s, p<0.05) and waves (3.2±1.2 vs. 0 events/min, p<0.05) were significantly higher in Pitx2+/- mice. This was also true for the frequency of transient inward currants activated by calcium waves (2.8±0.5 vs 1.2±0.5 events/min, p<0.05). The higher frequency was of spontaneous calcium release was likely due to a higher caffeine releasable sarcoplasmic reticulum calcium load in the Pitx2+/- mice (23.8±5.8 vs 14.4±1.9 amol/pF, p<0.05). Moreover, only myocytes from Pitx2+/- had spontaneous action potentials at a resting potential of −80 mV (0.5±0.4/min). At −60 mV the frequency of spontaneous action potentials was 5.3±1.8/min for Pitx2+/- and 0.4±1.2/min for Pitx2+/+ mice. Importantly, spontaneous after-potentials were also recorded in myocytes subjected to field stimulation, but only in myocytes from Pitx2+/- mice. These results were not unique to right atrial myocytes as the calcium spark frequency was also 3.9 fold higher in left atrial myocytes from Pitx2+/- mice (p<0.001).Together, these results demonstrate that Pitx2 insufficiency promote both spontaneous calcium release and spontaneous action potentials. Both of these features are hallmarks of myocytes from patients with atrial fibrillation, suggesting that Pitx2-mediated modulation of intracellular calcium handling plays an important role in electrophysiological processes associated with atrial fibrillation.
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Multiple Roles of Pitx2 in Cardiac Development and Disease
MDPI AG, 2017Co-Authors: Diego Franco, David Sedmera, Estefanía Lozano-velascoAbstract:Cardiac development is a complex morphogenetic process initiated as bilateral cardiogenic mesoderm is specified at both sides of the gastrulating embryo. Soon thereafter, these cardiogenic cells fuse at the embryonic midline configuring a symmetrical linear cardiac tube. Left/right bilateral asymmetry is first detected in the forming heart as the cardiac tube bends to the right, and subsequently, atrial and ventricular chambers develop. Molecular signals emanating from the node confer distinct left/right signalling pathways that ultimately lead to activation of the homeobox Transcription Factor Pitx2 in the left side of distinct embryonic organ anlagen, including the developing heart. Asymmetric expression of Pitx2 has therefore been reported during different cardiac developmental stages, and genetic deletion of Pitx2 provided evidence of key regulatory roles of this Transcription Factor during cardiogenesis and thus congenital heart diseases. More recently, impaired Pitx2 function has also been linked to arrhythmogenic processes, providing novel roles in the adult heart. In this manuscript, we provide a state-of-the-art review of the fundamental roles of Pitx2 during cardiogenesis, arrhythmogenesis and its contribution to congenital heart diseases
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A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate
2016Co-Authors: Estefanía Lozano-velasco, Diego Franco, Daniel Vallejo, Francisco J Esteban, Chris Doherty, Francisco Hernández-torres, Amelia AranegaAbstract:The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the Transcription Factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, andmiR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancingMyf5 satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine. The maintenance and repair of adult muscle tissue are directedby satellite cells. Quiescent satellite cells are activated by exer-cise or injury and enter the cell cycle to produce progenymyogenic precursor cells that undergo multiple rounds of division before entering terminal differentiation and fusing to multinucleated myofibers (1). Together with skeletal muscles, satellite cells origi-nate from cells of the segmented paraxial mesoderm known as somites. Somite formation starts at around embryonic day 7.75 (E7.75) in the mouse embryo and continues until the species
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Current Perspectives in Cardiac Laterality
MDPI AG, 2016Co-Authors: Marina Campione, Diego FrancoAbstract:The heart is the first organ to break symmetry in the developing embryo and onset of dextral looping is the first indication of this event. Looping is a complex process that progresses concomitantly to cardiac chamber differentiation and ultimately leads to the alignment of the cardiac regions in their final topology. Generation of cardiac asymmetry is crucial to ensuring proper form and consequent functionality of the heart, and therefore it is a highly regulated process. It has long been known that molecular left/right signals originate far before morphological asymmetry and therefore can direct it. The use of several animal models has led to the characterization of a complex regulatory network, which invariably converges on the Tgf-β signaling molecule Nodal and its downstream target, the homeobox Transcription Factor Pitx2. Here, we review current data on the cellular and molecular bases of cardiac looping and laterality, and discuss the contribution of Nodal and Pitx2 to these processes. A special emphasis will be given to the morphogenetic role of Pitx2 and to its modulation of Transcriptional and functional properties, which have also linked laterality to atrial fibrillation
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a Pitx2 microrna pathway modulates cell proliferation in myoblasts and skeletal muscle satellite cells and promotes their commitment to a myogenic cell fate
Molecular and Cellular Biology, 2015Co-Authors: Estefania Lozanovelasco, Diego Franco, Daniel Vallejo, Francisco J Esteban, Chris Doherty, Francisco Hernandeztorres, Amelia AranegaAbstract:The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the Transcription Factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5(+) satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine.
Amelia Aranega - One of the best experts on this subject based on the ideXlab platform.
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A Pitx2-MicroRNA Pathway Modulates Cell Proliferation in Myoblasts and Skeletal-Muscle Satellite Cells and Promotes Their Commitment to a Myogenic Cell Fate
2016Co-Authors: Estefanía Lozano-velasco, Diego Franco, Daniel Vallejo, Francisco J Esteban, Chris Doherty, Francisco Hernández-torres, Amelia AranegaAbstract:The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the Transcription Factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, andmiR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancingMyf5 satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine. The maintenance and repair of adult muscle tissue are directedby satellite cells. Quiescent satellite cells are activated by exer-cise or injury and enter the cell cycle to produce progenymyogenic precursor cells that undergo multiple rounds of division before entering terminal differentiation and fusing to multinucleated myofibers (1). Together with skeletal muscles, satellite cells origi-nate from cells of the segmented paraxial mesoderm known as somites. Somite formation starts at around embryonic day 7.75 (E7.75) in the mouse embryo and continues until the species
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a Pitx2 microrna pathway modulates cell proliferation in myoblasts and skeletal muscle satellite cells and promotes their commitment to a myogenic cell fate
Molecular and Cellular Biology, 2015Co-Authors: Estefania Lozanovelasco, Diego Franco, Daniel Vallejo, Francisco J Esteban, Chris Doherty, Francisco Hernandeztorres, Amelia AranegaAbstract:The acquisition of a proliferating-cell status from a quiescent state as well as the shift between proliferation and differentiation are key developmental steps in skeletal-muscle stem cells (satellite cells) to provide proper muscle regeneration. However, how satellite cell proliferation is regulated is not fully understood. Here, we report that the c-isoform of the Transcription Factor Pitx2 increases cell proliferation in myoblasts by downregulating microRNA 15b (miR-15b), miR-23b, miR-106b, and miR-503. This Pitx2c-microRNA (miRNA) pathway also regulates cell proliferation in early-activated satellite cells, enhancing Myf5(+) satellite cells and thereby promoting their commitment to a myogenic cell fate. This study reveals unknown functions of several miRNAs in myoblast and satellite cell behavior and thus may have future applications in regenerative medicine.
Philip J. Gage - One of the best experts on this subject based on the ideXlab platform.
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oculomotor nerve guidance and terminal branching requires interactions with differentiating extraocular muscles
Developmental Biology, 2021Co-Authors: Brielle Bjorke, Philip J. Gage, Katherine G Weller, Lauren E Jones, Eric G Robinson, Michelle Vesser, Lisheng Chen, Thomas W Gould, Grant S MastickAbstract:Abstract Muscle function is dependent on innervation by the correct motor nerves. Motor nerves are composed of motor axons which extend through peripheral tissues as a compact bundle, then diverge to create terminal nerve branches to specific muscle targets. As motor nerves approach their targets, they undergo a transition where the fasciculated nerve halts further growth then after a pause, the nerve later initiates branching to muscles. This transition point is potentially an intermediate target or guidepost to present specific cellular and molecular signals for navigation. Here we describe the navigation of the oculomotor nerve and its association with developing muscles in mouse embryos. We found that the oculomotor nerve initially grew to the eye three days prior to the appearance of any extraocular muscles. The oculomotor axons spread to form a plexus within a mass of cells, which included precursors of extraocular muscles and other orbital tissues and expressed the Transcription Factor Pitx2. The nerve growth paused in the plexus for more than two days, persisting during primary extraocular myogenesis, with a subsequent phase in which the nerve branched out to specific muscles. To test the functional significance of the nerve contact with Pitx2+ cells in the plexus, we used two strategies to genetically ablate Pitx2+ cells or muscle precursors early in nerve development. The first strategy used Myf5-Cre-mediated expression of diphtheria toxin A to ablate muscle precursors, leading to loss of extraocular muscles. The oculomotor axons navigated to the eye to form the main nerve, but subsequently largely failed to initiate terminal branches. The second strategy studied Pitx2 homozygous mutants, which have early apoptosis of Pitx2-expressing precursor cells, including precursors for extraocular muscles and other orbital tissues. Oculomotor nerve fibers also grew to the eye, but failed to stop to form the plexus, instead grew long ectopic projections. These results show that neither Pitx2 function nor Myf5-expressing cells are required for oculomotor nerve navigation to the eye. However, Pitx2 function is required for oculomotor axons to pause growth in the plexus, while Myf5-expressing cells are required for terminal branch initiation.
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AP-2b Is a Downstream Effector of Pitx2 Required to Specify Endothelium and Establish Angiogenic Privilege During Corneal Development
2020Co-Authors: Lisheng Chen, Vanessa Martino, Alan Dombkowski, Trevor Williams, Judith West-mays, Philip J. GageAbstract:Citation: Chen L, Martino V, Dombkowski A, Williams T, West-Mays J, Gage PJ. AP-2b is a downstream effector of Pitx2 required to specify endothelium and establish angiogenic privilege during corneal development. Invest Ophthalmol Vis Sci. 2016;57:107257: -108157: . DOI:10.1167 PURPOSE. The homeodomain Transcription Factor, Pitx2, is at the apex of a genetic pathway required for corneal development, but the critical effector genes regulated by the Pitx2 remain unknown. The purpose of this study was to discover and validate Pitx2-dependent mechanisms required for specifying cell lineages and establishing angiogenic privilege within the developing cornea. METHODS. Microarrays were used to compare gene expression in corneas isolated from temporal Pitx2 knockout embryos and control littermates. Quantitative RT-PCR and immunohistochemistry was used to further validate Tfap2b expression differences in Pitx2 knockout versus control corneas. In situ hybridization and protein immunohistochemistry were used to assay eyes of a Tfap2b allelic series of embryos to identify differentiated cellular lineages in the cornea, blood vessel endothelium, or lymphatic vessel endothelium. RESULTS. We show that Pitx2 is required for the expression of Tfap2b, encoding the AP-2b Transcription Factor, in the neural crest during corneal development. Markers of differentiated corneal epithelium and stroma are expressed in the absence of AP-2b. In contrast, markers of differentiated corneal endothelium are not expressed in the absence of AP-2b. Endomucin þ blood vessels are present throughout the developing corneal stroma in the absence of AP-2b, whereas LYVE1 þ lymphatic vessels are not found. CONCLUSIONS. The AP-2b Transcription Factor is an important effector of Pitx2 function during corneal development, required for differentiation of corneal endothelium and establishment of angiogenic privilege. Unlike Pitx2, AP-2b is not required for the early expression of available lineage specific markers for the corneal epithelium and stroma during embryogenesis, nor establishment of lymphangiogenic privilege. Therefore, additional Pitx2-dependent Factors likely regulate these latter processes during embryonic development. These results extend our understanding of the genetic mechanisms regulating cornea development
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mutation of foxc1 and Pitx2 induces cerebral small vessel disease
Journal of Clinical Investigation, 2014Co-Authors: Curtis R French, Philip J. Gage, Kathleen J. Millen, Sudha Seshadri, Anita L Destefano, Myriam Fornage, Corey R Arnold, Jonathan M Skarie, William B Dobyns, William H DietzAbstract:–/– Patients with cerebral small-vessel disease (CSVD) exhibit perturbed end-artery function and have an increased risk for stroke and age-related cognitive decline. Here, we used targeted genome-wide association (GWA) analysis and defined a CSVD locus adjacent to the forkhead Transcription Factor FOXC1. Moreover, we determined that the linked SNPs influence FOXC1 transcript levels and demonstrated that patients as young as 1 year of age with altered FOXC1 function exhibit CSVD. MRI analysis of patients with missense and nonsense mutations as well as FOXC1-encompassing segmental duplication and deletion revealed white matter hyperintensities, dilated perivascular spaces, and lacunar infarction. In a zebrafish model, overexpression or morpholino-induced suppression of foxc1 induced cerebral hemorrhage. Inhibition of foxc1 perturbed platelet-derived growth Factor (Pdgf) signaling, impairing neural crest migration and the recruitment of mural cells, which are essential for vascular stability. GWA analysis also linked the FOXC1-interacting Transcription Factor Pitx2 to CSVD, and both patients with Pitx2 mutations and murine Pitx2 –/– mutants displayed brain vascular phenotypes. Together, these results extend the genetic
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the homeodomain Transcription Factor Pitx2 is required for specifying correct cell fates and establishing angiogenic privilege in the developing cornea
Developmental Dynamics, 2014Co-Authors: Philip J. Gage, Chen Cha'o-kuang, Amanda L ZachariasAbstract:Background: Correct specification of cell lineages and establishing angiogenic privilege within the developing cornea are essential for normal vision but the mechanisms controlling these processes are poorly understood. Results: We show that the homeodomain Transcription Factor Pitx2 is expressed in mesenchymal cells of the developing and mature cornea and use a temporal gene knockout approach to demonstrate that Pitx2 is required for corneal morphogenesis and the specification of cell fates within the surface ectoderm and mesenchymal primordia. Pitx2 is also required to establish angiogenic privilege in the developing cornea. Further, the expression of Dkk2 and suppression of canonical Wnt signaling activity levels are key mechanisms by which Pitx2 specifies ocular surface ectoderm as cornea. In contrast, specifying the underlying mesenchyme to corneal fates and establishing angiogenic privilege in the cornea are less sensitive to DKK2 activity. Finally, the cellular expression patterns of FOXC2, PITX1, and BARX2 in Pitx2 and Dkk2 mutants suggest that these Transcription Factors may be involved in specifying cell fate and establishing angiogenic privilege within the corneal mesenchyme. However, they are unlikely to play a role in specifying cell fate within the corneal ectoderm. Conclusions: Together, these data provide important insights into the mechanisms regulating cornea development. Developmental Dynamics 243:1391–1400, 2014. © 2014 Wiley Periodicals, Inc.
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human prkc apoptosis wt1 regulator is a novel Pitx2 interacting protein that regulates Pitx2 Transcriptional activity in ocular cells
Journal of Biological Chemistry, 2009Co-Authors: Moulinath Acharya, Philip J. Gage, David J Lingenfelter, L Huang, Michael A WalterAbstract:Abstract Mutations in the homeobox Transcription Factor Pitx2 result in Axenfeld-Rieger syndrome (ARS), which is associated with anterior segment dysgenesis and an increased risk of glaucoma. To understand the pathogenesis of the defects resulting from Pitx2 mutations, it is essential to know the normal functions of Pitx2 and its interaction with the network of proteins in the eye. Yeast two-hybrid screening was performed using a cDNA library from a human trabecular meshwork primary cell line to detect novel Pitx2-interacting proteins and study their role in ARS pathogenesis. After screening of ∼1 × 106 clones, one putative interacting protein was identified named PRKC apoptosis WT1 regulator (PAWR). This interaction was further confirmed by retransformation assay in yeast cells as well as co-immunoprecipitation in ocular cells and nickel pulldown assay in vitro. PAWR is reportedly a proapoptotic protein capable of selectively inducing apoptosis primarily in cancer cells. Our analysis indicates that the homeodomain and the adjacent inhibitory domain in Pitx2 interact with the C-terminal leucine zipper domain of PAWR. Endogenous PAWR and Pitx2 were found to be located in the nucleus of ocular cells and to co-localize in the mesenchyme of the iridocorneal angle of the developing mouse eye, consistent with a role in the development of the anterior segment of the eye. PAWR was also found to inhibit Pitx2 Transcriptional activity in ocular cells. These data suggest PAWR is a novel Pitx2-interacting protein that regulates Pitx2 activity in ocular cells. This information sheds new light in understanding ARS and associated glaucoma pathogenesis.
Amanda L Zacharias - One of the best experts on this subject based on the ideXlab platform.
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the homeodomain Transcription Factor Pitx2 is required for specifying correct cell fates and establishing angiogenic privilege in the developing cornea
Developmental Dynamics, 2014Co-Authors: Philip J. Gage, Chen Cha'o-kuang, Amanda L ZachariasAbstract:Background: Correct specification of cell lineages and establishing angiogenic privilege within the developing cornea are essential for normal vision but the mechanisms controlling these processes are poorly understood. Results: We show that the homeodomain Transcription Factor Pitx2 is expressed in mesenchymal cells of the developing and mature cornea and use a temporal gene knockout approach to demonstrate that Pitx2 is required for corneal morphogenesis and the specification of cell fates within the surface ectoderm and mesenchymal primordia. Pitx2 is also required to establish angiogenic privilege in the developing cornea. Further, the expression of Dkk2 and suppression of canonical Wnt signaling activity levels are key mechanisms by which Pitx2 specifies ocular surface ectoderm as cornea. In contrast, specifying the underlying mesenchyme to corneal fates and establishing angiogenic privilege in the cornea are less sensitive to DKK2 activity. Finally, the cellular expression patterns of FOXC2, PITX1, and BARX2 in Pitx2 and Dkk2 mutants suggest that these Transcription Factors may be involved in specifying cell fate and establishing angiogenic privilege within the corneal mesenchyme. However, they are unlikely to play a role in specifying cell fate within the corneal ectoderm. Conclusions: Together, these data provide important insights into the mechanisms regulating cornea development. Developmental Dynamics 243:1391–1400, 2014. © 2014 Wiley Periodicals, Inc.
Joris R De Groot - One of the best experts on this subject based on the ideXlab platform.
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Pitx2 modulates atrial membrane potential and the antiarrhythmic effects of sodium channel blockers
Journal of the American College of Cardiology, 2016Co-Authors: Fahima Syeda, Andrew P Holmes, Samantha Tull, Stefan Michael Kuhlmann, Davor Pavlovic, Daniel Betney, Genna Riley, Jan P Kucera, Florian Jousset, Joris R De GrootAbstract:Background Antiarrhythmic drugs are widely used to treat patients with atrial fibrillation (AF), but the mechanisms conveying their variable effectiveness are not known. Recent data suggested that paired like homeodomain-2 Transcription Factor (Pitx2) might play an important role in regulating gene expression and electrical function of the adult left atrium (LA).