To determine the cause for the nonexchangeability of Rnd2 and Rnd3, we compared their activities in neuronal migration. Silencing Rnd2 and Rnd3 in side-by-side knockdown experiments resulted in migration defects of similar

severity ( Figure S3B) and silencing the two genes simultaneously resulted in a limited worsening of the migration defect, with a small increase in cell accumulation within the VZ/SVZ and concomitant decrease in the fraction of cells reaching the CP when compared with single knockdown experiments ( Figure S3B). Thus Rnd3 and Rnd2 are both required for the migration of cortical neurons and their individual functions are mostly distinct CHIR-99021 clinical trial and nonredundant. In agreement with this interpretation, the effects of Rnd2 and Rnd3 silencing on the morphology of migrating neurons were drastically different ( Figures 4A and 4B). Rnd3-silenced neurons that reached the CP presented aberrant morphologies, including a grossly enlarged leading process and multiple thin processes extending from the cell body and the leading process ( Figures 4A–4C; Movie S1). An excess number of primary processes were also observed in Rnd3-silenced cortical neurons in culture ( Figure 4D). Migration of neurons along

glial fibers in the CP involves successive phases of leading process extension AZD8055 mw and cell body translocation, during which the nucleus moves toward the centrosome located in a dilation of the leading process. Cell press The enlarged proximal leading process of Rnd3-silenced neurons suggested that translocation of the soma into the leading process may be impaired in these cells. Indeed, the average distance between the nucleus and the centrosome in neurons of the lower CP was markedly increased in Rnd3-silenced neurons (2.7 ± 0.4 μm) compared with control or Rnd2-silenced neurons (1.1 ± 0.2 μm and 1.4

± 0.3 μm, respectively; Figure 2E), suggesting that Rnd3 activity is required for nucleus-centrosome coupling in locomoting neurons in the CP (see also Movie S1). Rnd2-silenced neurons did not present this defect ( Figure 4E) and had a normally shaped leading process when they reached the CP ( Figure 4A), although most of them failed to leave the IZ where they accumulated with a multipolar morphology ( Figure 4B; Heng et al., 2008). Together, these data suggest that Rnd3 and Rnd2 are required during distinct phases of migration of cortical neurons and regulate different aspects of the migratory process. To understand the basis for the divergent functions of Rnd3 and Rnd2 in migrating neurons, we next characterized their downstream signaling pathways. Rnd3 has been shown to regulate cell morphology and migration in cultured fibroblasts and cancer cells by antagonizing RhoA ( Chardin, 2006 and Riento et al., 2005b). To determine whether Rnd3 also regulates RhoA signaling in the developing cortex, we measured RhoA activity in cortical cells by fluorescence resonance energy transfer (FRET) analysis. A FRET probe for RhoA ( Matthews et al.