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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.
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