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First published online 16 August 2006
doi: 10.1242/dev.02543

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Connexin 43-mediated modulation of polarized cell movement and the directional migration of cardiac neural crest cells

Laboratory of Developmental Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20850, USA.

View larger version (109K): [in a new window]   Fig. 1. CNC explants express neural crest markers. Immunostaining show expression of Crabp1 (A,B) and Sox10 (C,D), two neural crest markers, in CNCs emerging from postotic hindbrain neural tube explants derived from wild-type (A,C) and Cx431KO (B,D) mouse embryos. Scale bar: 50 µm.

View larger version (35K): [in a new window]   Fig. 2. Cx431 modulation of neural crest cell motility on fibronectin. The speed and directionality of cell locomotion was quantitatively assessed using time lapse images of CNCs explants from Cx431KO and CMV43 embryos plated on different fibronectin coating densities. Images were captured every 10 minutes over a 20-hour interval. As fibronectin density increased, wild-type and nontransgenic control CNCs (clear bars) show increased directionality, but decreased speed of cell locomotion (A,B). However, Cx431KO cells failed to achieve high directionality, while their speed of cell locomotion decreased more rapidly (A). By contrast, CMV43 neural crest cells exhibited higher directionality and higher speed of cell locomotion at 1 and 15 µg/ml fibronectin, respectively (B). Numbers in bars indicate the total number of cells analyzed. Error bars indicate the s.e.m. *P<0.05; **P<0.01; ***P<0.001, compared with wild type. #P<0.05; ##P<0.01; ###P<0.001, compared with hemi- or heterozygotes.

View larger version (36K): [in a new window]   Fig. 3. Cx431 modulation of cell protrusive activity. (A) Time lapse image stacks obtained from individual Cx431+/+, Cx431-/- and CMV43 CNCs captured at 5-minute intervals. Area of expansion or positive flow is indicated in red; area of contraction or negative flow is indicated in green. The image on top represents the cell at the last two frames, with underlying cell outlines corresponding to the preceding 10 timelapse imaging frames. (B,C) Cell protrusive activity in Cx431KO (B) and CMV43 (C) CNCs was quantitatively assessed by measuring positive (green) and negative (red) cytoplasmic flow. Roundness was also analyzed, which provided a measure of cell shape and was calculated using formula 100% x 4 (area/perimeter2), with the maximal roundness being 100% for a circle. The Cx431KO cells showed increased positive and negative flows, together with decreased roundness (B), while no change in positive/negative flow or roundness was seen for the CMV43 CNCs (C). *P<0.05 when compared with Cx431+/+ or nontransgenic controls. The numbers of cells analyzed are indicated in the bars.

View larger version (58K): [in a new window]   Fig. 4. Alterations in focal adhesion contacts in Cx431 KO and CMV43 CNCs. (A-C) CNC cells from wild-type (A), Cx431KO (B) and CMV43 (C) embryos were cultured on 15 µg/ml fibronectin matrix. Cells were double immunostained with antibodies against ß1 integrin (red) or vinculin (green). Cx431KO cells show reduced ß1 integrin and vinculin immunostaining (B). In CMV43 CNCs, the pattern of ß1 integrin and vinculin immunofluorescence is qualitatively changed, with the intensity of vinculin immunostaining showing a noticeable increase (C). Magnification is same in all panels. Scale bar: 50 µm. (D) To examine if ß1 integrin is expressed in CNCs, wild-type and CMV43 CNC explants were surface biotinylated, then cell extracts were made, followed by immunoprecipitation using a ß1 integrin antibody and western immunoblotting using stretpavidin-peroxidase. This yielded a 120 kDa band, the size expected for ß1 integrin. No difference was detected in the surface ß1 integrin expression level in the CMV43 and wild-type CNCs. (E,F) The mean area and mean intensity of vinculin immunofluorescence in CNCs cultured on 1, 15 and 50 µg/ml fibronectin matrix were quantitatively assessed. Cx431KO cells showed decreases in vinculin area and intensity (E), while CMV43 cells showed an increase in vinculin intensity (F). *P<0.05, **P<0.01; #P<0.0005. n/s, no significant difference, when compared with Cx431+/+ or nontransgenic cells.

View larger version (82K): [in a new window]   Fig. 5. Cx431KO CNCs show alteration in the actin cytoskeleton. (A,B) Rhodamine phalloidin staining showed parallel alignment of actin stress fibers in wild type CNCs (A), whereas in Cx431KO CNCs, stress fiber bundles were oriented in a polygonal array around the cell periphery (B). (C,D) Double immunostaining with rhodamine phalloidin (red) and vinculin (green) showed actin stress fibers typically terminated at focal adhesions in wild-type CNCs (C), but in Cx431KO cells (D), some actin stress fibers were not associated with focal adhesions (arrow). (E-I) Phase-contrast images show morphology of CNCs before (E) and after (F) cytochalasin D treatment, and 1 hour post-removal (G) of cytochalasin D. As CNCs re-establish their normal cell morphology, rhodamine phalloidin staining showed wild-type CNCs reformed parallel actin filament bundles (H), while Cx431KO CNCs reformed a multiangular ring of actin filaments around the cell periphery (I). Nuclei in A,B,H,I are delineated in blue using overlay of phase contrast images. Scale bars: 50 µm. Magnification is the same in A-D,H,I and in E-G.

View larger version (25K): [in a new window]   Fig. 6. Inhibition of CNC migration by ß1 integrin function blocking antibody. The motile behavior of CNC cells treated with ß1 integrin function blocking antibody was monitored by time lapse imaging and motion analysis, with images captured every 10 minutes. The change in directionality and speed of cell locomotion was compared with untreated cells and plotted as percent change in directionality (A) or speed (B). The wild-type CNCs show a reduction in speed and directionality within 1 hour of antibody treatment. The Cx431 KO and CMV43 CNCs showed a delayed response to the inhibitory effects of the antibody treatment. Error bars indicate s.e.m. *1 P<0.05, *2 P<0.01, *3 P<0.001 when compared with pre-antibody treatment; *4 P<0.05, *5 P<0.01 when compared with 1 hour of antibody treatment; *6 P<0.05 when compared with 2 hours of antibody treatment. Number of cells analyzed: 16, wild type; 19, Cx431KO; 20, CMV43.

View larger version (43K): [in a new window]   Fig. 7. Cx431 perturbation alters the response of CNCs to Sema3a. (A) Timelapse images of Cx431+/+, Cx431-/- and CMV43 CNCs treated with Sema3a. Images were captured at 1-minute intervals over 20 minutes, with area of expansion indicated by green (positive flow) and area of contraction indicated in red (negative flow). The image on top represents the cell at the last time frame, with underlying cell outlines corresponding to preceding timelapse frames. (B) Positive and negative flows were decreased in wild-type, Cx431KO and CMV43 CNCs after Sema3a treatment, but the cell roundness was increased only in the wild-type CNCs (B). Error bars indicate s.e.m. *P<0.05, when compared with cells without Sema3A of same genotype.

View larger version (92K): [in a new window]   Fig. 8. Cx431 colocalization with actin filaments and actin binding proteins. CNCs were double immunostained for Cx431 (green, A,D,G,J,M,P) and either vinculin (B), ezrin (H), IQGAP-1 (K), -actinin (N) or drebrin (Q) in red. F-actin (E) was stained using rhodamine phalloidin. In the merged images (C,F,I,L,O,R), regions of Cx431 colocalization with actin or these various actin-associated proteins can be observed. These regions of colocalization were mostly found at areas of cell-cell contact and along cell processes (see white arrowhead in insets). Boxed regions are magnified twofold in the bottom insets. Scale bar: 50 µm. (S) NIH3T3 cell extracts were immunoprecipitated with a Cx431 antibody and western immunoblotted with a vinculin antibody. A band of the size expected for vinculin was observed in the Cx431 immunoprecipitate and in the total lysate. (T) NIH3T3 cell extracts were immunoprecipitated with an IQGAP-1 antibody and western immunoblotted with a Cx431 antibody. A band of the size expected for Cx431 was observed in the IQGAP-1 immunoprecipitate and in the total lysate.

View larger version (28K): [in a new window]   Fig. 9. Quantitative analysis of dye coupling in CNCs. (A) Intracellular microelectrode impalement and iontophoretic injection of 6-carboxyfluorescein in a CNC explant culture plated on 15 µg/ml fibronectin. (B) Extensive dye spread can be seen in the same explant after 2 minutes of iontophoretic dye injection. The microelectrode was removed at this 2-minute timepoint. (C) More extensive dye spread can be seen in the same explant after an additional 2 minutes to allow for continued spread of the injected dye. (D). To assess the precise extent of dye spread from the impaled cell (0) to primary (1), secondary (2) and tertiary neighbors (3), the dark-field fluorescence image in C was merged with the cell outlines obtained by tracing the DIC image of the same area. Shown in red are cells in the secondary or tertiary cell layers that did not contain the injected dye. Scale bars: 100 µm.

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