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Connexin 43 regulates epicardial cell polarity and migration in coronary vascular development.
Rhee DY, Zhao XQ, Francis RJ, Huang GY, Mably JD, Lo CW.
???displayArticle.abstract??? Connexin 43 knockout (Cx43 KO) mice exhibit conotruncal malformations and coronary artery defects. We observed epicardial blisters in the Cx43 KO hearts that suggest defects in epicardial epithelial-mesenchymal transformation (EMT), a process that generates coronary vascular progenitors. Analysis using a three-dimensional collagen gel invasion assay showed that Cx43 KO epicardial cells are less invasive and that, unlike wild-type epicardial cells, they fail to organize into thin vessel-like projections. Examination of Cx43 KO hearts using Wt1 as an epicardial marker revealed a disorganized pattern of epicardial cell infiltration. Time-lapse imaging and motion analysis using epicardial explants showed a defect in directional cell migration. This was associated with changes in the actin/tubulin cytoskeleton. A defect in cell polarity was indicated by a failure of the microtubule-organizing center to align with the direction of cell migration. Forced expression of Cx43 constructs in epicardial explants showed the Cx43 tubulin-binding domain is required for Cx43 modulation of cell polarity and cell motility. Pecam staining revealed early defects in remodeling of the primitive coronary vascular plexuses in the Cx43 KO heart. Together, these findings suggest an early defect in coronary vascular development arising from a global perturbation of the cytoarchitecture of the cell. Consistent with this, we found aberrant myocardialization of the outflow tract, a process also known to be EMT dependent. Together, these findings suggest cardiac defects in the Cx43 KO mice arise from the disruption of cell polarity, a process that may be dependent on Cx43-tubulin interactions.
Fig. 2. Cx43 KO epicardial cells exhibit a defect in actin cytoskeleton organization. (A-D) The actin cytoskeleton and focal adhesion contacts in wild-type and Cx43 KO epicardial cells from E11.5 epicardial explant cultures were visualized with rhodamine-conjugated phalloidin (A,C, red) and vinculin antibody (B,D, green). Wild-type cells (A,B) show actin stress fibers aligned with the direction of cell migration (indicted by arrow in A), and dense actin filaments delineate the regions of cell-cell contact. By contrast, in the KO cells, the actin stress fibers were often not oriented with the direction of cell migration (indicated by arrow in C), and the density of actin filaments was reduced at regions of cell-cell contact. In these same cells, vinculin immunostaining showed prominent adhesion plaques in the wild-type epicardial cells (B). By contrast, KO epicardial cells have finer adhesion plaques (D). Scale bar in A: 10 μm.
Fig. 3. Altered distribution of ZO-1 in Cx43 KO epicardial explants. (A,B) E11.5 epicardial explant cultures were immunostained with ZO-1 (green) antibody. Strong cell surface localization was observed at regions of cell-cell contract in wild-type epicardial cells (A). In Cx43 KO explants (B), ZO-1 localization at the cell surface was reduced concomitant with ZO-1 redistribution to the cytoplasm. The ZO-1 staining pattern in the KO epicardial explant showed many regions of apparent discontinuity in the epithelial sheet (arrowheads). Scale bar in A: 30 μm.
Fig. 4. Epicardial cell invasion in 3D collagen gel matrix. E11.5 epicardial cells were explanted on a 3D collagen gel matrix and then stained with rhodamine-conjugated phalloidin and DAPI to visualize epicardial cell invasion into the matrix. (A,B) Wild-type explants (A) exhibited organized outgrowths with long extended projections into the collagen gel matrix, but this was not observed in the Cx43 KO epicardial cells (B). (C-F) Confocal imaging of these collagen gel explants (shown along the x/y plane in C and D) was used to generate 3D reconstructions for measurement of cell invasion depth along the z-axis (E,F). (G) Measurements along the z-axes showed a significant decrease in the depth of cell invasion in the KO explants compared with wild type (n=6 explants per genotype, 120 cells measured for each). Scale bars: 250 μm in A,B; 50 μm in C; 75 μm in D.
Fig. 7. Defect in the specification of cell polarity in Cx43 KO epicardial cells. (A-D) Epicardial cells from E11.5 explant cultures were immunostained with antibodies to α-tubulin (A,B), or γ-tubulin and the Golgi GM130 marker (C,D) to visualize the MTOC. α-Tubulin staining exhibited an intensely stained crescent-shaped structure that corresponds to the MTOC. In wild-type cells, most of the MTOC are forward facing, i.e. situated in the leading edge of the cell (white arrow indicates the direction of cell migration). The microtubules were also observed to align with the direction of cell migration. By contrast, in the Cx43 KO explants, the MTOC and the microtubule cytoskeleton were often not aligned with the direction of cell migration (B). A similar result was obtained when γ-tubulin and GM130 antibodies were used to track the positioning of the MTOC in wild-type (C) and KO epicardial cells (D). The quantitative analysis is shown in the bar graphs to the right. Red and blue staining in A,B and C,D, respectively, corresponds to DAPI staining. Scale bar: 50 μm.
Barker,
Increased association of ZO-1 with connexin43 during remodeling of cardiac gap junctions.
2002, Pubmed
Barker,
Increased association of ZO-1 with connexin43 during remodeling of cardiac gap junctions.
2002,
Pubmed Clauss,
Patterning of coronary arteries in wildtype and connexin43 knockout mice.
2006,
Pubmed Dai,
Cx43 mediates TGF-beta signaling through competitive Smads binding to microtubules.
2007,
Pubmed Dettman,
Common epicardial origin of coronary vascular smooth muscle, perivascular fibroblasts, and intermyocardial fibroblasts in the avian heart.
1998,
Pubmed Duffy,
Formation of the gap junction nexus: binding partners for connexins.
2002,
Pubmed Ewart,
Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene.
1997,
Pubmed Giepmans,
Connexin-43 interactions with ZO-1 and alpha- and beta-tubulin.
2001,
Pubmed Giepmans,
Gap junction protein connexin-43 interacts directly with microtubules.
2001,
Pubmed Giepmans,
The gap junction protein connexin43 interacts with the second PDZ domain of the zona occludens-1 protein.
,
Pubmed Gittenberger-de Groot,
Epicardium-derived cells contribute a novel population to the myocardial wall and the atrioventricular cushions.
1998,
Pubmed Goldberg,
Gap junctions between cells expressing connexin 43 or 32 show inverse permselectivity to adenosine and ATP.
2002,
Pubmed Guo,
Gap junction-microtubule associations in rat alveolar epithelial cells.
2003,
Pubmed Hood,
Coronary artery development in the chick: origin and deployment of smooth muscle cells, and the effects of neural crest ablation.
1992,
Pubmed Huang,
Alteration in connexin 43 gap junction gene dosage impairs conotruncal heart development.
1998,
Pubmed Huang,
Gap junction-mediated cell-cell communication modulates mouse neural crest migration.
1998,
Pubmed Imamura,
Functional domains of alpha-catenin required for the strong state of cadherin-based cell adhesion.
1999,
Pubmed Itoh,
Involvement of ZO-1 in cadherin-based cell adhesion through its direct binding to alpha catenin and actin filaments.
1997,
Pubmed Johnson,
Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules.
2002,
Pubmed Jordan,
Trafficking, assembly, and function of a connexin43-green fluorescent protein chimera in live mammalian cells.
1999,
Pubmed Kanno,
The role of myocardial gap junctions in electrical conduction and arrhythmogenesis.
2001,
Pubmed Kupfer,
Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound.
1982,
Pubmed Kupfer,
Polarization of the Golgi apparatus and the microtubule-organizing center within cloned natural killer cells bound to their targets.
1983,
Pubmed Kwee,
Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice.
1995,
Pubmed Lauf,
Dynamic trafficking and delivery of connexons to the plasma membrane and accretion to gap junctions in living cells.
2002,
Pubmed Li,
An essential role for connexin43 gap junctions in mouse coronary artery development.
2002,
Pubmed Li,
Alterations in Cx43 and OB-cadherin affect breast cancer cell metastatic potential.
2008,
Pubmed Magdalena,
Microtubule involvement in NIH 3T3 Golgi and MTOC polarity establishment.
2003,
Pubmed Männer,
Does the subepicardial mesenchyme contribute myocardioblasts to the myocardium of the chick embryo heart? A quail-chick chimera study tracing the fate of the epicardial primordium.
1999,
Pubmed Mikawa,
Pericardial mesoderm generates a population of coronary smooth muscle cells migrating into the heart along with ingrowth of the epicardial organ.
1996,
Pubmed Montcouquiol,
[Planar polarity in mammals: similarity and divergence with Drosophila Melanosgaster].
2007,
Pubmed Muñoz-Chápuli,
Cellular precursors of the coronary arteries.
2002,
Pubmed Nambara,
Role of the cytoplasmic loop domain of Cx43 in its intracellular localization and function: possible interaction with cadherin.
2007,
Pubmed Nobes,
Rho GTPases control polarity, protrusion, and adhesion during cell movement.
1999,
Pubmed Omori,
Gap junction proteins connexin32 and connexin43 partially acquire growth-suppressive function in HeLa cells by deletion of their C-terminal tails.
1999,
Pubmed Pérez-Pomares,
Experimental studies on the spatiotemporal expression of WT1 and RALDH2 in the embryonic avian heart: a model for the regulation of myocardial and valvuloseptal development by epicardially derived cells (EPDCs).
2002,
Pubmed Phillips,
Vangl2 acts via RhoA signaling to regulate polarized cell movements during development of the proximal outflow tract.
2005,
Pubmed Phillips,
Non-cell-autonomous roles for the planar cell polarity gene Vangl2 in development of the coronary circulation.
2008,
Pubmed Phillips,
Disruption of planar cell polarity signaling results in congenital heart defects and cardiomyopathy attributable to early cardiomyocyte disorganization.
2007,
Pubmed Poelmann,
Development of the cardiac coronary vascular endothelium, studied with antiendothelial antibodies, in chicken-quail chimeras.
1993,
Pubmed Polette,
Beta-catenin and ZO-1: shuttle molecules involved in tumor invasion-associated epithelial-mesenchymal transition processes.
2007,
Pubmed Prochnow,
Connexons and cell adhesion: a romantic phase.
2008,
Pubmed Reaume,
Cardiac malformation in neonatal mice lacking connexin43.
1995,
Pubmed Runyan,
Invasion of mesenchyme into three-dimensional collagen gels: a regional and temporal analysis of interaction in embryonic heart tissue.
1983,
Pubmed Saburi,
Loss of Fat4 disrupts PCP signaling and oriented cell division and leads to cystic kidney disease.
2008,
Pubmed Saez,
Plasma membrane channels formed by connexins: their regulation and functions.
2003,
Pubmed Scemes,
Modulation of astrocyte P2Y1 receptors by the carboxyl terminal domain of the gap junction protein Cx43.
2008,
Pubmed Shaw,
Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions.
2007,
Pubmed Soll,
The use of computers in understanding how animal cells crawl.
1995,
Pubmed Stites,
Phosphorylation of the Dictyostelium myosin II heavy chain is necessary for maintaining cellular polarity and suppressing turning during chemotaxis.
1998,
Pubmed Sullivan,
Expression of a connexin 43/beta-galactosidase fusion protein inhibits gap junctional communication in NIH3T3 cells.
1995,
Pubmed Thomas,
Role of cytoskeletal elements in the recruitment of Cx43-GFP and Cx26-YFP into gap junctions.
2001,
Pubmed Virágh,
The origin of the epicardium and the embryonic myocardial circulation in the mouse.
1981,
Pubmed Vrancken Peeters,
Smooth muscle cells and fibroblasts of the coronary arteries derive from epithelial-mesenchymal transformation of the epicardium.
1999,
Pubmed Waldo,
Association of the cardiac neural crest with development of the coronary arteries in the chick embryo.
1994,
Pubmed Walker,
Connexin43 deficiency causes dysregulation of coronary vasculogenesis.
2005,
Pubmed Wei,
Connexins and cell signaling in development and disease.
2004,
Pubmed Wei,
Connexin43 associated with an N-cadherin-containing multiprotein complex is required for gap junction formation in NIH3T3 cells.
2005,
Pubmed Xu,
Modulation of mouse neural crest cell motility by N-cadherin and connexin 43 gap junctions.
2001,
Pubmed Xu,
Connexin 43-mediated modulation of polarized cell movement and the directional migration of cardiac neural crest cells.
2006,
Pubmed Yang,
Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development.
1995,
Pubmed Zhou,
Epicardial progenitors contribute to the cardiomyocyte lineage in the developing heart.
2008,
Pubmed
