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Cell coupling and Cx43 expression in embryonic mouse neural progenitor cells

¹ Institut Pasteur, Unité de Neurovirologie et Régénération du Système Nerveux, 75015 Paris, France
² Département de Morphologie, Centre Médical Universitaire, 1211 Geneva, Switzerland

View larger version (109K): [in a new window]   Fig. 1. Adherent neuropsheres form layers of migrating cells. Phase-contrast micrographs show that, 30 minutes after adhesion of neurospheres on coated coverslips (A), few cells have started to migrate out of the sphere. 24 hours after adhesion (B), outgrowing cells formed layers (arrow) surrounding the sphere (dotted circle). After 7 days (C), the number of cells decreased in the sphere (dotted circle), which was flattened, and increased in the outgrowth. Bar, 150 µm (A); 300 µm (B,C).

View larger version (79K): [in a new window]   Fig. 2. Dye coupling is present between cells in the neurosphere and between differentiating astrocytes. Lucifer Yellow (LY) was microinjected by iontophoresis into single cells. Fluorescence micrographs were taken 5 minutes after injection; insets show the corresponding phase-contrast views of the same fields. Dye coupling was strong between cells located in the neurosphere center (A) and between cells identified as astrocytes (B). By contrast, no LY diffusion to adjacent cells was observed when an oligodendrocyte (C) or a neuron (D) was microinjected. Bar, 10 µm.

View larger version (111K): [in a new window]   Fig. 3. Identification of coupled astrocytes. After microinjection experiments, neurospheres were fixed and labeled as indicated in the Materials and Methods. A typical cluster of dye-coupled cells (green) showed positive staining for the astrocytic protein GFAP (red). Bar, 20 µm.

View larger version (126K): [in a new window]   Fig. 4. Cell proliferation in adherent neurospheres. Neurospheres were allowed to adhere for 7 days and then incubated for 24 hours in the presence of 10 µM BrdU. Cells were fixed and processed for double-labeling with anti-BrdU (green) and anti-GFAP (red) antibodies. Note that most BrdU-positive cells were concentrated within the sphere, whereas only a few of them were found in the outgrowth. Bar, 100 µm.

View larger version (87K): [in a new window]   Fig. 5. A blockade of gap junctions decreases the viability of neural progenitors. Single cell suspensions of striatal progenitors were grown for 3 days in the presence of either the uncoupling agent 18-ß-glycyrrhetinic acid (ßGA) or its inactive analog glycyrrhyzic acid (GZA). The size of neurospheres was reduced by ßGA (C,D) in comparison with that of GZA-treated ones (A,B). This effect was reversible (rev), as the size of floating neurospheres pre-treated with ßGA (G-H) was similar to that of GZA-treated cultures (E-F) following an additional 4 day recovery period, as described in the Materials and Methods. Bar, 300 µM (A-D); 780 µM (E-H).

View larger version (117K): [in a new window]   Fig. 6. The uncoupling agent ßGA perturbs the morphology of differentiating cells. Floating neurospheres were allowed to differentiate either in the presence of ßGA or DMSO, which was used as solvent of the drug. Both the density and morphology of cells migrating out of the spheres were altered by ßGA (E-H), whereas DMSO alone (A-D) had no effect. Astrocytes were thinner and elongated (F), and oligodendrocytes (G) exhibited reduced cell arborizations compared with cells grown in the presence of DMSO (B,C, respectively). Neurons (H) were mainly round and devoid of processes. In a series of independent experiments, ßGA was added after neurospheres had been allowed to adhere for three days and had formed an outgrowth of differentiated cells(I-L). Under these conditions, ßGA did not alter either the morphology or the viability of the three cell types. Bar, 80 µm (A-C, E-G, I-K); 25 µm (D,H,L).

View larger version (15K): [in a new window]   Fig. 7. ßGA alters the viability of both neural progenitors and differentiating cells. Viable cells of either floating (A) or adherent (B) neurospheres were quantified colorimetrically by the MTT assay (see Materials and Methods) following three days in the presence of the specified drugs. Data were normalized to the values measured under control conditions. The gap junction inhibitor ßGA caused a significant reduction of cell viability at all concentrations tested, whereas neither the inactive analog GZA nor solvent (DMSO/ethanol, DE) affected the total number of viable cells. Results are shown as means±s.e.m. of three independent experiments. Statistical significance was analyzed using the unpaired Student's t-test (*P<0.01).

View larger version (51K): [in a new window]   Fig. 8. Neural progenitors and differentiated astrocytes express Cx43. Single cell suspensions of striatal progenitors were expanded for 10 days in the presence of EGF to form floating neurospheres, which were fixed, sectioned and immunolabeled with a monoclonal antibody raised against Cx43 (B). The image corresponds to the merging of 10 planes acquired by confocal microscopy. Virtually all cells in the neurosphere were positive for Cx43. The corresponding Nomarski view is shown in A. Bar, 75 µm. (C) Neurospheres were allowed to differentiate for 8 days and were double-labeled with a polyclonal antibody against Cx43 (red) and a monoclonal antibody anti-GFAP (green). Cx43 was detected in GFAP-positive cells. Bar, 10 µm.

View larger version (90K): [in a new window]   Fig. 9. Time course of Cx43 and GFAP expression in differentiating neurospheres. Floating neurospheres, expanded for 10 days with EGF, were transferred onto coated coverslips and allowed to differentiate for 30 minutes (A,B), 3 hours (C,D) and 8 days (E,F). Double immunostaining was performed using a rabbit polyclonal antibody against Cx43 (red) and a mouse monoclonal against GFAP (green). Pictures were taken from three different neurospheres and are representative of three independent experiments for each time point. Images in A, C and E result from the merging of 14 confocal planes, whereas single plane views of the same neurospheres are shown in B, D and F. At each adhesion time, Cx43 was expressed by cells located within the sphere. Cx43 expression increased with time in cells migrating out of the sphere. GFAP expression was detectable only in a few cells at 30 minutes of adhesion but was strongly enhanced in the outgrowing layers at 3 hours and 8 days of adhesion. Bar, 10 µm in A-B; 30 µm in C-F.

View larger version (13K): [in a new window]   Fig. 10. Cx43 phosphorylation is increased in adherent neurospheres. Cell lysates were prepared from either floating (grown for 10 days) or adherent (8 days) neurospheres. Equal amounts of proteins were incubated overnight at 37°C in the presence of either digestion buffer (lanes 1 and 4), alkaline phosphatase (lanes 2 and 5) or alkaline phosphatase plus an excess of phosphatase inhibitors (lanes 3 and 6). Samples were separated by SDS-gel electrophoresis and immunoblotted with either a rabbit polyclonal (A) or a mouse monoclonal (B) anti-Cx43 antibody. Both antibodies reacted with Cx43 from floating neurospheres, and the apparent electrophoretic mobility was not perturbed by alkaline phosphatase treatment (lanes 1-3). In adherent neurospheres, a broader band was detected when the blot was probed with the polyclonal antibody (panel A, lane 4). Following exposure to phosphatase, Cx43 shifted to faster migrating forms (panel A, lane 5). The monoclonal anti-Cx43 antibody, which recognizes non-phosphorylated Cx43, detected Cx43 species only when the lysates were treated with alkaline phosphatase (panel B, lane 5). It should be noted that twice the amount of protein was loaded in B. This blot is representative of three others from two independent experiments.

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