The developing brain is a very interesting place. One fascinating aspect is the migration of embryonic neurons from their birthplace to their adult position. Embryonic neurons in the neocortex migrate to their appropriate position by crawling along fibers which are created by radial glial cells. Radial glia also act as stem cells, replicating and differentiating into new neurons. The molecular interactions between infant neurons and radial glial cells, until recently, was relatively unknown. Recent research from the Institute of Regenerative Medicine shows that gap junctions play a prominent role in the migration of neurons.

More details after the jump.

Several methods of action have been proposed but gap junctions appeared the most likely. Gap junctions are large hemi-channels that punch through the plasma membrane. Hemi-channels link to form an aqueous pore between the two cells. Two gap junctions expressed in the developing cortex include Cx26 and Cx43. Previous research has shown that radial glial cells are connected to each other with gap junctions and are required for a proper cell cycle. It was theorized that these same gap junctions may play and important part in neuron-glial interactions as well. The two gap junctions do not appear to colocalize together. Both types of gap junctions are expressed in migrating neurons and radial glial fibers , suggesting they play an important part in migration.

To test this theory, the research team selected short hairpin RNS (shRNA) constructs that significantly reduced the expression of Cx26 and Cx43 gap junctions (Cx26-shRNA and Cx43-shRNA, respectively). Another shRNA (ctr-shRNA) was selected which did not knockdown the expression of the gap junctions. This shRNA was used as the control in the test. The team injected the shRNA constructs into embryonic neocortical cells and noticed significant redistribution of cells as compared to the control. There was a large decrease in mobility of neural stem cells and many areas were under-represented when compared to the control.

Radial glial cells use gap junctions themselves for structure. The team wanted to verify that the disruption in neuron migration was due to impaired neuron-glial cell communication rather than a problem in the structure the neurons were traveling over. After all, it is hard to drive over a bridge with a big hole in the middle. The team confirmed visually that the structure of the radial glial scaffolding was intact and normal.

To further determine that the problem was in neural-glial interaction, the team injected individual shRNA-expressing cells into wild-type brains. The control cells (expressing ctr-shRNA) were able to migrate as normal but the Cx26-shRNA and Cx43-shRNA had problems. The knockout shRNAs were capable of engrafting to the structure present in the wildtype substrates but showed reduced ability to migrate. This showed that something inherent in the gap junctions is necessary for neural migration.

It was confirmed that neurons were exiting the cell cycle and differentiating as normal. Apoptosis levels were also normal. This is important because it shows that the migration problems was not a result of premature death. Lastly, it was shown that the knockout didn’t overtly interfere with other adhesion proteins required for normal cell life. Cells with decreased Cx43 expression adhered to control substrates but not Cx43 expressing substrates, meaning it was still expressing regular adhesion proteins.

Now that the team had shown that gap junctions were critical to neuronal migration, they wanted to determine what aspect of gap junctions was responsible for it. Gap junctions can serve three roles in a cell. If two cells have gap junctions linked together, they can allow communication between two cells by exchanging small particles. If the cell has a gap junction exposed to the extracellular environment without a partner, it can be used to dump substrates outside the cell. Lastly, it can provide an adhesive contact between cells.

To test if the the channel property of two gap junctions was responsible, a mutation in the gap junctions was made. These mutated gap junctions adhered to each other as normal to form a pore but the mutation blocked the flow of substrates through the channel. It was shown that these mutants, despite blocking all flow of substrate between the two cells, rescued migration of neural cells along the fibers. This strongly suggests that migration is not reliant on any transmittance of substrate from radial glial cells to neurons.

It was previously found that neocortical gap junctions mediate Ca²⁺ waves by releasing ATP into the extracellular environment. Ca²⁺ waves have been shown to be involved in neural migration, so it was theorized perhaps the release of ATP from gap junctions stimulates migration. To test this, Ca²⁺ waves were inhibited with an antagonist. No problems in migration were seen, clearly showing that Ca²⁺ waves, while important in other areas of migration, was not relevant in the pathway being studied.

Lastly, the team tested the adhesive properties of gap junctions. A mutated gap junction was created that did not make adhesions and therefor could not create proper gap junctions. Unlike the other tests, there was noticeable migration problems. This test combined with the previous tests strongly shows that the adhesive properties of gap junctions is required for neuronal migration.

The two gap junctions looked at in this study were found to localize on the leading processes of a migrating neuron. The neurons looked at tended to start migration with two leading processes. One of these will dominate (usually by finding and attaching to a radial glial fiber) and the other will disappear. Neurons that lack the Cx43 and Cx26 gap junctions were unable to stabilize their processes on a fiber and thus unable to move forward. The data gathered from this study clearly shows that Cx43/Cx26 gap junctions are required for developing neocortex neuronal migration because of their adhesive function. Cx26 tended to play a role in nuclear translocation while Cx43 helped in branch stabilization.

Until now, research has not explored the role of adhesive properties in gap junctions rather than activity in its channel. The team suggests that other problems that rely on gap junctions, such as migration of gliobastoma in brain tumors, may be caused by similar gap junction adhesion. They also suggest that neurological disorders such as Charcot-Marie-Tooth syndrome (caused by mutations in Cx32) might be the result of faulty adhesion rather than faulty communication between gap junctions.

References

Elias L, Wang D, Kriegstein A. Gap junction adhesion is necessary for radial migration in the neocortex. Nature (2007) 448:901-907. 10.1038/nature06063