pRaichu-1298x, which carries the C-terminal region of RhoA including the CAAX box, delivers the probe preferentially to intracellular membrane compartments, whereas pRaichu-1293x, which carries the C terminus of K-Ras, delivers the probe preferentially to the plasma membrane

pRaichu-1298x, which carries the C-terminal region of RhoA including the CAAX box, delivers the probe preferentially to intracellular membrane compartments, whereas pRaichu-1293x, which carries the C terminus of K-Ras, delivers the probe preferentially to the plasma membrane. (C) FRET analysis of RhoA activity in cortical cells in?vivo, 1?day after electroporation of pRaichu-1293x WP1066 together with or shRNA. it is unclear whether this activity reflects a genuine role in cortical neuron migration and the downstream mechanisms involved are unknown. During development of the cerebral cortex, excitatory projection neurons generated in the ventricular zone (VZ) and subventricular zone (SVZ) of the dorsal telencephalon migrate radially through the intermediate zone (IZ) to reach the superficial layers of the cortical plate (CP). Distinct phases of neuronal migration and correlated morphologies of migrating neurons can be distinguished (LoTurco and Bai, 2006). Neurons initiate migration in the VZ with a bipolar morphology, they become transiently multipolar in the SVZ and IZ, and they convert back to a bipolar morphology to enter the CP. Bipolar neurons migrate along radial glial fibers by using a mode of migration termed locomotion, which involves a reiterative succession of steps affecting different cellular domains. Neurons extend their leading process along radial glia fibers and translocate their nucleus and perinuclear region into the proximal leading process, a process known as nucleokinesis, which is WP1066 followed by retraction of the trailing process, resulting in overall movement of the neuron (Marn et?al., 2006). The different steps of neuronal migration involve extensive reorganization of the cytoskeleton and, not surprisingly, Rho GTPases, which control many aspects of cytoskeleton dynamics (Heasman and Ridley, 2008), have been implicated in migration of different types of neurons (Govek et?al., 2005; Heasman and Ridley, 2008; Marn et?al., 2006). Rac1 is required for the formation of the leading process in cortical neurons (Kawauchi et?al., 2003; Konno et?al., 2005), while Cdc42 is important for nuclear movements in postmitotic cerebellar granule neurons (Kholmanskikh et?al., 2006), and RhoA activity is required for nucleokinesis and organization of the cytoskeleton at the rear end of migrating precerebellar neurons (Causeret et?al., 2004). Although many pathways are known to control the activity of Rho, Rac, and Cdc42 in nonneuronal cells, much less is known of how the activity of these small GTPases is controlled in migrating neurons. The atypical Rho protein Rnd3/Rho8/RhoE is an important regulator of migration of fibroblasts and tumor cells (Chardin, 2006; Guasch WP1066 et?al., 1998; Klein and Aplin, 2009; Nobes et?al., 1998) that acts by inhibiting RhoA through stimulation of the Rho GTPase-activating protein p190RhoGAP (Wennerberg et?al., 2003), and/or inhibition of the activity of ROCKI, one of the main effectors of RhoA (Riento et?al., WP1066 2003). Rnd3 has been shown to induce neurite outgrowth in pheochromocytoma PC12 cells, but its role in neuronal migration has not been examined (Talens-Visconti et?al., 2010). A related protein, Rnd2/Rho7/RhoN, has been shown to promote the radial migration of cortical neurons (Heng et?al., 2008; Nakamura et?al., 2006) and to inhibit neurite growth and induce neurite branching in PC12 cells (Fujita et?al., 2002; Tanaka et?al., 2006), but the mechanisms mediating Rnd2 activity in neurons remain PTGER2 unclear. Rnd2 and Rnd3 belong to the small Rnd family of atypical Rho proteins that lack intrinsic GTPase activity and are therefore constitutively bound to GTP (Chardin, 2006). Rnd proteins are thought to be regulated at the level of their expression, phosphorylation, and subcellular localization (Madigan et?al., 2009; Riento et?al., 2005a). We have previously shown that the proneural protein Neurog2 promotes the migration of nascent cortical neurons through induction of expression as part of an extensive subtype-specific transcriptional program controlling cortical neurogenesis (Heng et?al., 2008). In this study, we have further investigated how the cell behavior of radial migration of cortical neurons is regulated in the context of a global developmental program. We show that another proneural factor expressed in the embryonic cortex, Ascl1, promotes neuronal migration through regulation of Is a Direct Transcriptional Target of Ascl1 We began this study by asking whether the proneural transcription factor Ascl1, which has been shown to enhance cell migration when overexpressed in cultured cortical cells (Ge et?al., 2006), is required for neuronal migration during development of the cerebral cortex. We examined the consequence of acute loss of function in the embryonic cortex by introducing an expression construct encoding the Cre recombinase in the cortex of embryos carrying a conditional mutant allele of Ascl1 (mice resulted in a significant reduction of the radial migration of electroporated cells at E17.5 when compared with electroporation of only GFP (Figure?1A), demonstrating that is required for proper neuronal migration in the embryonic cortex. We next asked whether transcripts are normally present in the telencephalon of mutant embryos, whereas they are clearly depleted in mutants (Heng et?al., 2008; Figure?S1D), suggesting that Ascl1 does not regulate expression. To identify alternative mechanisms through which Ascl1 promotes migration, we searched for candidate target genes of Ascl1 that might be involved in regulating cell migration (Gohlke et?al., 2008; Figure?S1E). By using gene expression microarrays, we found that family of small GTP-binding proteins.