Quantification of the proportion of GFP-labeled cells (CreGFP-electroporated cells in RhoAfl/+ animals or GFP-electroporated check details cells in RhoAfl/fl embryos were used as a control; Figure 4D, yellow bars) confirmed not only the efficient neuronal migration but even revealed an apparently faster migration of RhoA-depleted neurons as a significantly higher proportion of GFP+ cells was located in the upper most bin of five equally bins, corresponding to the cortical plate (CP), compared to controls ( Figure 4D, blue bars). These experiments therefore suggest that RhoA-depleted neurons did not fail to migrate but rather migrated faster than control cells. Some RhoA-depleted cells had migrated even beyond the CP forming ectopic
clusters within and beyond layer 1 in brains analyzed at E19, i.e., 5 days after electroporation ( Figures 4E and 4F), reminiscent of the type II cobblestone lissencephaly observed in the cerebral cortex of cKO mice and described previously. To test the possibility that RhoA may indeed learn more slow down migration and release this break in its absence, we electroporated a spontaneously activated (“fast-cycling”) mutant of RhoA (RhoA∗) and quantified the position of GFP+ cells in the cerebral cortex. Indeed, consistent with this scenario, we detected a significant increase of GFP-labeled cells in the lower layers 3 days after electroporating
the fast-cycling RhoA mutant construct ( Figures 4C, 4C′, and 4D, pink bars), suggesting a delay in migration in the condition of activated RhoA. Even though these migrating neurons because also had a normal polarized morphology, consistent with a normal migration, it would still be possible that RhoA-deficient neurons reach their final position but in a very different or disturbed migration compared to normal. We therefore directly monitored the migration
of electroporated cells by live imaging in slices. E13 Cre-electroporated cortices were sliced and sections were imaged 2 days after transfection for approximately 9 hr to examine the movement of migrating cells (Movie S1; Figures 5A–5D). All cells imaged performed a normal radial migration moving basally and directed by a single leading process, as shown by the traces of tracked cells in Figures 5A–5D. However, despite the early reduction of RhoA protein in electroporated regions, the remnant levels may still be sufficient to allow for migration of these neurons. To examine migration of neurons lacking RhoA protein entirely, we employed transplantation experiments with cells from E14 cKO cortices which had completely lost RhoA protein by E12. E14 cells were dissociated and labeled with cell tracker green prior to transplantation into isochronic WT cortices (Figures 6A and 6B). Similar to control cells also RhoA−/− cells had often reached the IZ or CP 3 days after transplantation ( Figures 6C–6E). Thus, neurons still migrate fairly normal and reach the CP also in the complete absence of RhoA.