ARTICLES Gap junction adhesion is necessary for radial migration in the neocortex Laura A. B. Elias 1,2 , Doris D. Wang 1,2 & Arnold R. Kriegstein 2 Radial glia, the neuronal stem cells of the embryonic cerebral cortex, reside deep within the developing brain and extend radial fibres to the pial surface, along which embryonic neurons migrate to reach the cortical plate. Here we show that the gap junction subunits connexin 26 (Cx26) and connexin 43 (Cx43) are expressed at the contact points between radial fibres and migrating neurons, and acute downregulation of Cx26 or Cx43 impairs the migration of neurons to the cortical plate. Unexpectedly, gap junctions do not mediate neuronal migration by acting in the classical manner to provide an aqueous channel for cell–cell communication. Instead, gap junctions provide dynamic adhesive contacts that interact with the internal cytoskeleton to enable leading process stabilization along radial fibres as well as the subsequent translocation of the nucleus. These results indicate that gap junction adhesions are necessary for glial-guided neuronal migration, raising the possibility that the adhesive properties of gap junctions may have an important role in other physiological processes and diseases associated with gap junction function. During neocortical brain development, radial glia, which reside in the ventricular zone and extend a fibre to the pial surface, serve two purposes: (1) as stem cells of the developing cortex, giving rise to neurons 1,2 , and (2) as guides along which the neurons migrate to reach the correct lamina of the cortical plate, where they will become pyramidal cells in the adult cortex 3–5 . Interstitial junctions containing filamentous material have been found between migrating neurons and radial fibres, suggesting a close association between the two cell types 6–8 . The molecules that mediate the critical interaction between the radial glial fibre and the migrating neuron remain largely unknown. Astrotactin has been described as an adhesion molecule in the cere- bellar cortex 9,10 , but has not been implicated in neocortical develop- ment. Neuregulin and its soluble form, glial growth factor, mediate bi-directional signalling between the radial fibre and the migrating neuron, and are important for radial glial maintenance and neuronal migration 11 . a 3 b 1 integrin expressed in migrating neurons is involved in glial recognition, and a V integrin expressed in radial glia is involved in neuronal adhesion 12 . The expression of gap junctions in radial fibres and migrating neurons, together with the detection of gap junctions between nestin 1 radial glial fibres and nestin 2 cells by electron microscopy, suggests the possibility that gap junctions play a part in neuron–glial interactions and neuronal migration 13 . This hypothesis is supported by the change in the distribution of bromodeoxyuridine (BrdU) pulse-labelled cells in the Cx43 genetic knockout (Cx43KO) 14 . Furthermore, strong evidence links Cx43 to migration in neural crest cells and gliomas; however, the mechanisms are not fully understood 15–18 . Gap junctions are large-diameter channels made up of two hemi- channels—each composed of six connexin subunits—on opposing membranes that join through hydrophobic interactions and form an aqueous pore between the cytoplasm of two adjacent cells 19 . The gap junction subunits expressed in the developing cortex include Cx26 (also known as Gjb2), a b1 connexin family member, and Cx43 (also known as Gja1), an a1 connexin family member 20 . There is strong evidence that radial glia are coupled electrically and chem- ically to each other by gap junction channels during embryonic development 21 . Functionally, it has been suggested that gap junctions have a role in radial glial cell cycle regulation because phar- macological block of gap junctions, or Ca 21 waves mediated by gap junction hemichannels, inhibits the cell cycle 22,23 . However, the possible role of gap junctions in cortical migration remains largely unexplored. Gap junction expression at contact points The gap junction subunits Cx26 and Cx43 are expressed in the deve- loping rat neocortex. Whereas Cx26 is evenly distributed from the ventricular zone through the intermediate zone to the cortical plate, Cx43 is highly expressed at the ventricular surface and its levels are reduced in the cortical plate (Fig. 1a, b, e, f). Cx26 and Cx43 puncta do not colocalize with each other (Supplementary Fig. 1), because they belong to separate families that do not make heterotypic junc- tions or heteromeric hemichannels 24 . The expression of both con- nexins outside of the ventricular zone proliferative region indicates that gap junctions may regulate functions additional to radial glial proliferation. To address this possibility, we determined the pattern of Cx26 and Cx43 expression with respect to b-III tubulin 1 migrat- ing neurons and vimentin 1 radial glial fibres. Both gap junction subunits are expressed in migrating neurons and along radial fibres. In fact, Cx26 and Cx43 are highly localized in neurons to the regions of contact with radial fibres, consistent with the idea that gap junc- tions may have a role in mediating communication between migrat- ing neurons and their radial guides (Fig. 1, and Supplementary Movies 1, 2). Cx26 and Cx43 are necessary for migration To investigate the role of gap junctions in cortical development, we developed acute loss-of-function manipulations for each connexin using RNA interference. Short hairpin RNA (shRNA) constructs that produced significant knockdown of rat Cx26 or Cx43 by western blot in Cos-7 monkey cells were selected (Cx26-shRNA or Cx43-shRNA, respectively), and it was confirmed that the same hairpin sequences with three point mutations (Ctrl-shRNA) failed to produce signi- ficant knockdown (Supplementary Fig. 2a, b). We confirmed that 1 Neuroscience Graduate Program, 2 Institute for Regeneration Medicine, University of California San Francisco, 513 Parnassus Avenue, San Francisco, California 94143, USA. Vol 448 | 23 August 2007 | doi:10.1038/nature06063 901 Nature ©2007 Publishing Group