Connexin 43 mediates the tangential to radial migratory switch in ventrally derived cortical interneurons

Abstract

The adult cerebral cortex is composed of excitatory and inhibitory neurons that arise from progenitor cells in disparate proliferative regions in the developing brain and follow different migratory paths. Excitatory pyramidal neurons originate near the ventricle and migrate radially to their position in the cortical plate along radial glial fibers. On the other hand, inhibitory interneurons arise in the ventral telencephalon and migrate tangentially to enter the developing cortex before migrating radially to reach their correct laminar position. Gap junction adhesion has been shown to play an important mechanistic role in the radial migration of excitatory neurons. We asked whether a similar mechanism governs the tangential or radial migration of inhibitory interneurons. Using short hairpin RNA knockdown of Connexin 43 (Cx43) and Cx26 together with rescue experiments, we found that gap junctions are dispensable for the tangential migration of interneurons, but that Cx43 plays a role in the switch from tangential to radial migration that allows interneurons to enter the cortical plate and find their correct laminar position. Moreover this action is dependent on the adhesive properties and the C terminus of Cx43 but not the Cx43 channel. Thus, the radial phase of interneuron migration resembles that of excitatory neuron migration in terms of dependence on Cx43 adhesion. Furthermore, gap junctions between migrating interneurons and radial processes were observed by electron microscopy. These findings provide mechanistic and structural support for a gap junction-mediated interaction between migrating interneurons and radial glia during the switch from tangential to radial migration.

Figure 1.

Gap junctions observed by electron microscopy between migrating interneurons and radial glia in the cortical MZ. A, A′, Gap junction (arrow) between an interneuron process (IN) and radial glial process (*). B, B′, Gap junction (arrow) between an interneuron cell body (IN) and a radial glial process (*). Scale bars: A, 1 μm; B, 2 μm; A′, B′, 50 nm.

Figure 2.

Electroporation of Cx43-shRNA reduces the proportion of cells that switch from tangential to radial migration. A, The MGE of coronal slice cultures was electroporated with control plasmid (pLLox3.7) or Cx43-shRNA (GFP⁺ cells) at E15.5. Time-lapse images were acquired at 48, 72, and 96 h after electroporation. Images of the dorsal cortex show that Cx43-shRNA-expressing cells have a reduction in radial orientation and an increase in tangential orientation, resulting in impairment of the ability of cells to migrate radially into the cortex. B, Quantification of the shRNA knockdown-mediated cell orientation (left) and laminar positioning (right) effect. The percentage of radially oriented cells in the neocortex was significantly reduced by Cx43-shRNA expression at 48, 72, and 96 h after electroporation (number of cells per condition indicated on each bar) (Fisher's exact test, pLLox3.7 vs Cx43-shRNA, 48 h, p = 0.0078; 72 h, p = 0.0025; 96 h, p < 0.0001) (left). The laminar positioning of cells was normal at 48 h, but by 96 h there was a significant increase in the fraction of Cx43-shRNA-expressing cells localized in the outer one-third of the cortex adjacent to the cortical pial surface (t test, pLLox3.7 vs Cx43-shRNA, 48 h, p = 0.8662; 72 h, p = 0.0665; 96 h, p = 0.0037) (right).

Figure 3.

Both Cx43 adhesion and the CT are necessary for the switch from tangential to radial migration. A, Images of slice cultures at 72 h after electroporation expressing Cx43-shRNA along with a Cx43 rescue construct. B, Cx43CM (function: channel, adhesion, CT) and Cx43CMT154A (function: adhesion, CT) significantly increased the percentage of radially oriented cells when compared to EYFP at 72 and 96 h after coelectroporation with Cx43-shRNA (number of cells per condition indicated on each bar) (Fisher's exact test, Cx43CM, 72 h, p = 0.0316, 96 h, p = 0.0358; Cx43CMT154A, 72 h, p = 0.0045, 96 h, p = 0.0129). Cx43CMT154A 1-245 (function: adhesion), Cx43CMC61S (function: hemichannel, CT), or Cx43CT (function: CT) did not rescue the orientation defect.

Figure 4.

Overexpressing the Cx43CT alone has a dominant-negative effect, thus inhibiting the tangential to radial switch at 72 and 96 h, but does not affect cell branching patterns. A, Images of slice cultures 96 h after electroporation with EYFP or Cx43CT+EYFP. B, Compared to EYFP, Cx43CT significantly decreases the percentage of radially oriented cells at 72 and 96 h (number of cells per condition indicated on each bar) (Fisher's exact test, 72 h, p = 0.0174; 96 h, p = 0.0006) (left). At 96 h, compared to EYFP, there is a developing trend for Cx43CT expression to increase the fraction of cells found in the outer one-third of the cortex (t test, p = 0.1429) (right). C, Images of migrating interneurons in the dorsal cortex at 72 h after electroporation. D, Cx43-shRNA-expressing cells display a significantly higher number of primary and total branches (t test, pLLox3.7 vs Cx43-shRNA, primary, p = 0.0349; total, p = 0.0340). Cx26-shRNA and the Cx43CT do not affect branching patterns.