Involvement of the cytoplasmic C-terminal domain of connexin43 in neuronal migration.

During brain development, young neurons closely associate with radial glial while migrating from the ventricular zone (VZ) to the cortical plate (CP) of the neocortex. It has been shown previously that gap junctions are needed for this migration to occur properly, but the precise mechanism responsible is still in question. Here, we used Cre recombinase, driven by the nestin promoter, to conditionally knock-out a floxed coding DNA of the connexin43 (Cx43) gene in mice. Radial glia in the VZ normally express connexin43. They undergo divisions that produce neurons and astrocytes and serve as migratory guides for the daughter cells that they produce. Based on histological analysis, we suggest that removing Cx43 from radial glia alters the normal lamination of the mouse neocortex. To monitor newborn neurons during development, we introduced a plasmid containing green fluorescent protein driven by a neuronal (Talpha1 tubulin) promoter into the embryonic neocortex using in utero electroporation. The transfected migrating neurons remain in the VZ/intermediate zone (IZ) of the Cx43 conditional knock-out (Cx43cKO) animals, whereas in Cx43(fl/fl) mice, neurons migrate through the IZ into the CP, indicating that deletion of Cx43 from nestin-positive cells disrupts neuronal migration. We were able to rescue migration of Cx43cKO neurons by electroporating a cytomegalovirus-Cx43 expression plasmid into the embryonic cortex. In contrast, a C-terminal truncated form of Cx43 failed to rescue neuronal migration. In addition, Cx43(K258stop) mice, in which Cx43 lacks the last 125 amino acid residues of the cytoplasmic C-terminal domain, gave results similar to those seen with the Cx43cKO mice. This study illustrates that deletion of the C-terminal domain of Cx43 alters neuronal migration in the neocortex.

Expression of connexins in embryonic mouse neocortical development.

During embryonic development, young neurons migrate from the ventricular zone to the cortical plate of the cerebral cortex. Disturbances in this neuronal migration have been associated with numerous diseases such as mental retardation, double cortex, Down syndrome, and epilepsy. One possible cause of these neuropathologies is an aberration in normal gap junctional communication. At least 20 connexin (Cx) genes encode gap junction proteins in mice and humans. A proper understanding of the role of specific connexins in the developing brain requires the characterization of their spatial and temporal pattern of expression. In the current study we performed all the experiments on mouse developing cortex at embryonic days (E) 14, 16, and 18, timepoints that are highly active with regard to cortical development. Using reverse transcription-polymerase chain reaction, Western blot analysis, and immunohistochemistry, we found that among the family of gap junction proteins, Cx26, Cx36, Cx37, Cx43, and Cx45 were expressed in the developing cortex of mice, Cx30 and Cx32 were absent, while Cx40 was expressed at a very low level. Our results demonstrate that Cx26 and Cx37 were evenly distributed in the cortical layers of developing brain, while Cx36 and Cx43 were more abundant in the ventricular zone and cortical plate. Cx45 distribution appeared to be more abundant at E18 compared to the other timepoints (E14 and E16). Thus, the present study provides identification and the distribution pattern for Cxs associated with cortical development during normal neuronal migration.