In contrast, expression of DN-nectin3 or DN-afadin caused electro

In contrast, expression of DN-nectin3 or DN-afadin caused electroporated cells to accumulate

near the IZ (Figures 2E and 2F), indicating that nectin3 and afadin act in neurons, at least in part, to regulate glia-independent somal translocation. We next determined the mechanism by which nectin3 and afadin regulate radial migration. We reasoned that the two proteins might help to anchor the leading processes of neurons in the MZ. We therefore evaluated neuronal morphology following perturbation of nectin3 or afadin function by knockdown and dominant-negative approaches, which selleck gave similar results. Although neurons largely failed to migrate into the CP following perturbation of nectin3 or afadin, they still properly polarized the Golgi apparatus ahead of the nucleus (Figure 3A) and also developed stereotypical polarized morphologies characterized by leading processes (Figures 3B–3D). At 2–3 days after electroporation, leading processes that

extended toward or even into the MZ were observed in both control neurons and neurons expressing shRNAs against nectin3 or afadin (Figures 3B and 3C). A small decrease in the number of branches was observed after 3 days in the case of afadin shRNA electroporation, suggesting onset of leading-process retraction. However, only control neurons had their cell bodies located close to the MZ, indicative of somal translocation. Cell bodies in the knockdown experiments failed to translocate toward the MZ (Figures 3B and 3C) and remained near the IZ and lower CP, as nonelectroporated cells bypassed them to expand the CP. This CP expansion initially caused the leading processes of affected neurons to appear

longer than those of controls neurons below 2–3 days after electroporation (Figures 3B and 3C), but many of these processes were subsequently retracted by 4 days after electroporation (Figures 2C and 2F). Additionally, whereas the leading processes of control neurons extensively branched in the MZ, no such branching was observed after nectin3 or afadin knockdown (Figure 3D). Together, these data indicate that nectin3 and afadin are not required for neuronal polarization or initial process extension, but are important for leading-process anchorage and arborization in the MZ and subsequent somal translocation. To directly determine whether nectin3 and afadin are required for somal translocation, we carried out time-lapse imaging experiments. Neurons from E13.5 animals were electroporated with control, nectin3, or afadin shRNAs, and neocortical slice cultures were prepared at E15.5. As reported (Franco et al., 2011), control neurons translocated their cell bodies along their leading processes toward the MZ (Figure 3E). In contrast, neurons expressing shRNAs for afadin or nectin3 extended leading processes but failed to undergo somal translocation (Figure 3E).

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