Figure 4.

Rnd3 and Rnd2 Are Required during Different Phases of Migration

(A) Morphology of electroporated cells in the lCP. Rnd3-silenced neurons displayed an enlarged leading process (arrowhead) and multiple thin processes emanating from the cell body and leading process (arrows) not seen in Rnd2-silenced neurons.

(B) Quantification of the morphology of electroporated cells in the different zones of the cortex. The percentages represent the proportion of multipolar cells (i.e., cells exhibiting more than two primary processes) in each zone. n > 500 cells from three different brains; Student's t test; ^∗p < 0.05, ^∗∗∗p < 0.001 compared to control shRNA.

(C) Quantification of the number of branches emanating from the leading process. Mean ± SEM; n = 19–21 cells; one-way ANOVA followed by a Fisher's PLSD post hoc test; ^∗∗∗p < 0.001 compared to control shRNA; +++p < 0.001 compared to Rnd2 shRNA.

(D) Quantification of the number and average length of primary neurites in dissociated cortical neuron cultures established after ex vivo Rnd2 or Rnd3 silencing. Rnd2 and Rnd3 silencing both resulted in an increased number of primary processes in cultured cortical cells, but the processes were significantly longer than normal in Rnd2-silenced neurons and shorter in Rnd3-silenced neurons. The analysis was performed after two DIV by using ImageJ software. Mean ± SEM; n = 53–62 cells; one-way ANOVA followed by a Fisher's PLSD post hoc test; ^∗p < 0.05, ^∗∗p < 0.01 compared to control shRNA; +++p < 0.001 compared to Rnd2 shRNA.

(E) Analysis of the distance between centrosome and nucleus in cortical neurons coelectroporated with shRNA-RFP constructs and pClG2-Centrin2-Venus. The nucleus was labeled with TO-PRO-3. Mean ± SEM; n = 41 cells in all conditions; one-way ANOVA followed by a Fisher's PLSD post hoc test; ^∗∗∗p < 0.001 compared to control shRNA; +++p < 0.001 compared to Rnd2 shRNA. Scale bars represent 10 μm (A) and 5 μm (E). See also Figure S3 and Movie S1.