Rac in Radial Migration
Conditional knockout of Rac1 in the forebrain, as well as in utero electroporation studies in embryonic mice, demonstrate a requirement for Rac, its regulators, and effectors, in radial migration. Deletion of Rac1 in the telencephalic ventricular zone (VZ), using Foxg1-Cre mice, prevents cortical neurons from migrating to superficial layers of the neocortex by delaying the onset or reducing the speed of migration, rather than inhibiting it entirely (Chen et al., 2007). Neural precursors electroporated with either constitutively active (CA) Rac1V12 or dominant negative (DN) Rac1N17 exhibit retarded migration and a failure to form a leading process, suggesting that fine regulation of Rac activity levels and cycling of the GTPase are required for morphological polarization and migration (Konno et al.,2005; Kawauchi et al.,2003). Notably, the effects of DN Rac1N17 on radial migration are more severe than those of the Foxg1-Cre knockout and may reflect the ability of the DN mutant to bind/sequestor regulators utilized by other Rho GTPases and signaling pathways. A very recent report provides evidence that migration defects caused by loss of Rac1 in Foxg1-Cre mice may be due, at least in part, to defects in radial glial organization resulting from an inability to anchor their pial endfeet in the basement membrane (Leone et al.,2010).
A role for Rac in radial migration is further supported by a number of studies showing involvement of Rac regulators in this process (Fig. 7). These regulators include GEFs, such as STEF, Tiam1, P-Rex1, and Vav3. STEF and Tiam1 are Rac specific GEFs detectable in regions of the brain where neuronal migration and neurite outgrowth occur (Ehler et al.,1997; Yoshizawa etal.,2002), and they are both required for neurite formation in neuronal cell lines and primary neurons (Leeuwen et al.,1997; Matsuo et al.,2002, 2003). In the developing neocortex, STEF and Tiam1 are present in cells in the intermediate zone (IZ) and cortical plate (CP) (Yoshizawa et al.,2002; Kawauchi et al.,2003), and in utero electroporation of neural progenitors with DN STEF/Tiam causes cells to stall in the IZ without affecting their differentiation (Kawauchi et al.,2003). A study in cerebellar GCPs shows that Tiam1 and Rac act downstream of BDNF/TrkB signaling to mediate BDNF-induced directional migration, which involves polarized endocytosis of BDNF and TrkB in vitro and in cerebellar cortical slices (Zhou et al.,2007).
Figure 7. Rho GTPase signaling involved in radial migration. Some of the key molecules implicated in Rho GTPase signaling that play a role in radial migration, and potential interactors, are shown here (see text for details).
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P-Rex1 is another Rac-specific GEF that is expressed in the IZ and lower CP of the developing neocortex. Its expression is slightly more restricted than that of STEF and Tiam1, and it is expressed in Purkinje cells and possibly Bergmann glia of the developing and adult cerebellum. In migrating neurons in the cortical IZ, P-Rex1 protein localizes in the leading process and adjacent cytoplasmic region, and electroporation of DN P-Rex1 that lacks the Dbl-homology domain into embryonic cortex causes neuronal precursors to stall in the IZ (Yoshizawa et al.,2005).
Vav family members are Rac/Rho GEFs that play a role in axon guidance in retinal neurons and in Purkinje cell spinogenesis (Luo et al.,1996; Cowan et al.,2005). Vav3 is expressed at high levels in Purkinje cells and GCPs of the cerebellum and regulates Purkinje cell dendritogenesis, the timely migration of GCPs from the EGL to the IGL, and the survival of mature granule neurons (Quevedo et al.,2010).
Other molecules that act upstream of Rac in radial migration include two kinases, Phosphatidylinositol (PI) 3-kinase and Cdk5. PI 3-kinase produces PI 3,4,5-trisphosphate (PI[3,4,5]P3) and regulates cell polarization and migration via Rac, Cdc42, and/or Akt (protein kinase B) (Fukata et al.,2003). In utero electroporation of CA and DN PI 3-kinase mutants into neural progenitors in neocortical slices markedly inhibits radial migration. Suppression of Rac1 and Cdc42 activity by a PI 3-kinase inhibitor suggests that PI 3-kinase regulates radial migration through these GTPases (Konno et al.,2005).
Cyclin-dependent kinase 5 (Cdk5) is a neuron specific cyclin-dependent kinase regulated by subunits p35 and p39, which can complex with activated, GTP-bound Rac through p35, resulting in downregulation of the kinase activity of the Rac/Cdc42 effector Pak (Nikolic et al.,1998). Mutations in Cdk5 and p35 both cause an inversion of cortical layers (Ohshima et al.,1996; Chae et al.,1997; Gilmore etal.,1998; Ko et al.,2001; Ohshima et al.,2007). Introduction of DN Cdk5 via in utero electroporation arrests cells in the IZ (Kawauchi et al.,2003;2006; Nguyen etal.,2006), and Cdk5 deficient neurons fail to transition from the multipolar to the bipolar stage, form a leading process and translocate (Ohshima et al.,2007). p35 deficiency causes improper neuronal–glial interaction and aberrant branched migration instead of somal translocation (Gupta et al.,2003). Cdk5 may regulate Rac through the phosphorylation of Neurabin-1, a neuronal-specific F-actin-binding protein. Neurabin-1 negatively regulates Rac1 activity in the presence of Cdk5/p35, and both overexpression and knockdown of Neurabin-1 by in utero electroporation perturbs radial migration, with prominent effects on the leading process (Causeret et al.,2007). Neurabin-II may also negatively regulate Rac signaling by antagonizing the effects of JNK (c-jun N-terminal kinase), a member of the mitogen-activated protein kinase (MAPK) family known to act downstream of Rac (Davis,2000). Neurabin II promotes the dephosphorylation of JNK-mediated phosphorylation sites on DCX through protein phosphatase 1 (PP1; see JNK paragraph below for more information on JNK and DCX) (Shmueli et al.,2006; Tsukada etal.,2006). In addition to Neurabin, Cdk5 also targets both Reelin (RELN) and Lis1-related signaling proteins, and actin and MT regulatory molecules, including DCX, Ndel1, DAB1, and focal adhesion kinase (Niethammer et al.,2000; Sasaki etal.,2000; Keshvara et al.,2002; Xie et al.,2003; Tanaka et al.,2004b). Thus, Cdk5 may coordinately regulate different signaling pathways, and the actin and MT cytoskeletons, to effect radial migration.
Potential regulation of Rac downstream of the RELN/DAB1 signaling pathway may also occur during radial migration. When DAB1 is tyrosine phosphorylated, it provides a scaffold for signaling complexes, including Crk and CrkL adaptors, and CrkII links phosphorylated DAB1 to the Rac GEF DOCK180/DOCK1 (Ballif et al.,2004; Chen et al.,2004; Huang et al.,2004). Expression of phosphorylated DAB1 interferes with CrkII-dependent cell migration of Nara Bladder Tumor II (NBT-II) cells, and a loss-of-function mutation in myoblast city (mbc), the DOCK180 homolog in Drosophila, rescues the rough-eye phenotype in Drosophila caused by exogenous expression of phosphorylated mouse DAB1 (Chen et al.,2004), suggesting that DAB1 tyrosine phoshorylation may downregulate Rac activity. The absence of DAB1 Crk/CrkL binding sites, or Crk and CrkL deficiency, causes defects in cortical layering and loss of activity of the Crk-regulated Ras family GEF C3G (RapGEF1, Grf2) (Sanada et al.,2004; Park and Curran,2008; Feng and Cooper,2009). C3G-deficient mouse embryos also fail to split the preplate, form a cortical plate, and migrate, resulting in an arrest of neurons in the multipolar state and defects in migration (Voss et al.,2008). Thus, a likely scenario is that DAB1, Crk-DOCK180, and Rac1 as a complex contribute to radial migration.
Molecules that transduce Rac signaling also play a part in radial migration and include regulators of both the actin and MT cytoskeletons (Fig. 7). Actin regulators that act downstream of Rac that play a role in radial migration include, Pak, Ena/VASP, Abi2, and Filamin A. The Pak (p21-activated kinase) family of serine/threonine kinases, which includes Pak1-6, can be activated by, and serves as an effector for, both Rac and Cdc42 (Jaffer and Chernoff,2002; Bokoch,2003). Recent studies also reveal a role for Pak in cortical radial migration. Pak1 expression is high in migrating cortical neurons, and both CA and DN Pak1 mutants, introduced into the developing neocortex by in utero electroporation, perturb their morphology and radial migration. Overexpression of hyperactivated Pak1 causes neurons to arrest in the IZ with short processes that curve or branch and bear small protrusions. Neurons that reach the CP have broader leading processes with extensive lamellipodia. Loss of Pak1 disrupts the morphology of migrating neurons, which subsequently accumulate in the IZ and deep cortical layers. Neurons in the IZ elaborate disorganized extensions and broad lamellipodial protrusions that surround the soma, and those migrating in the CP have a shorter leading process. Unexpectedly, neurons with reduced Pak1 expression aberrantly enter into the marginal zone, suggesting over-migration, perhaps due to an inability to dissociate from radial glia (Causeret etal.,2009). Pak 1-3 may also cooperate with the cell adhesion molecule close homologue of L1 (CHL1) to regulate morphological development of the leading process/apical dendrite of embryonic cortical neurons (Demyanenko et al.,2010). Thus, Pak appears to play a major role in leading process formation during radial migration.
Members of the Ena/Vasp family [Mena (mammalian enabled), VASP, and EVL] control the elongation of unbranched actin filaments. They are localized in regions of dynamic actin reorganization, such as in the lamellipodium at the leading edge of motile cells (Reinhard et al.,1992; Gertler etal.,1996). In primary neurons, Mena is concentrated in the distal tips of growth cone filopodia and regulates axon guidance (Lanier et al.,1999). In developing cortical neurons, Ena/VASP proteins are highly expressed in IZ neurons and CP neurons bordering Reelin-expressing Cajal–Retzius cells. Loss of Ena/VASP function causes neuronal ectopias, alters intralayer positioning in the CP, and blocks axon fiber tract formation. The cortical fiber tract defects result from a failure in neurite initiation, which are preceded by a failure to form bundled actin filaments and filopodia (Goh et al.,2002; Kwiatkowski et al.,2007).
The Abl-interactor (Abi) family of adaptor proteins bind Abl tyrosine kinases (Dai and Pendergast,1995; Shi et al.,1995) and modulate Rac activity by acting both downstream and upstream of this GTPase. Abi1 and Abi2 bind a WAVE-interacting complex downstream of Rac to regulate Arp2/3-dependent actin polymerization (Eden et al.,2002; Soderling et al.,2002; Kunda et al.,2003; Rogers etal.,2003; Echarri et al.,2004; Innocenti et al.,2004; Steffen et al.,2004), and Abi1 complexes with Eps8 and Sos GEF to directly increase Rac activity and regulate actin dynamics (Scita et al.,1999, 2001; Innocenti et al.,2002, 2004). A role for Abi2 in neuronal migration has been reported. Abi2 is highly expressed in the brain and developing cortex and is prominent in the marginal zone (MZ) and along the border of the lateral ventricles, where it colocalizes with β-catenin. Although cortical layers appear to form normally, pyramidal cells are not radially aligned in adult abi2 null mice. Downregulation of Abi expression by RNA interference in epithelial cells suggests that these defects could arise from impaired adherens junction formation and downregulation of the WAVE actin-nucleation promoting factor (Grove etal.,2004).
Filamin-A is an actin binding protein that can bind Rac1 (Marti et al.,1997; Ohta et al.,1999; Stossel et al.,2001; Zhou et al.,2010), the Rac GEF Trio (Bellanger et al.,2000), and the Rac GAP FilGAP (Ohta etal.,2006), as well as positively regulate the Rac effector Pak (Vadlamudi et al.,2002; Maceyka et al.,2008). Mutations in the Filamin A gene, FLNa, cause the cortical malformation periventricular heterotopia (PH) that results from a failure in neuronal migration (Fox et al.,1998; Feng and Walsh,2004; Sarkisian et al.,2008). Filamin-A expression is high in the IZ above the subventricular zone (SVZ) and in the CP of the developing cortex (Fox et al.,1998; Nagano et al.,2002; Sato and Nagano,2005). Expression of a Filamin A mutant lacking the actin-binding domain arrests the migration of postmitotic neurons in the IZ of the mouse neocortex and impairs acquisition of a bipolar morphology in the SVZ/IZ, whereas Filamin A overexpression, achieved by downregulating the Filamin A-interacting protein that induces Filamin A degradation (FILIP), increases the number of bipolar cells (Nagano et al.,2004; Sato and Nagano,2005). These findings suggest that Filamin A is enriched in postmitotic, migratory neurons and that its ability to crosslink actin is required for its function in migration. In agreement with these data, FLNa mutations that cause PH are often truncations or disruptions of the actin-binding domain (Fox et al.,1998; Feng and Walsh,2004; Sarkisian et al.,2008). Filamin-A is also regulated by MEKK4/MAP3K4 (Sarkisian et al.,2006), a Rac1/Cdc42 binding protein and upstream MAPKKK for JNK (Davis,2000). Similar to mutations in FLNa, loss of MEKK4 causes PHs (Sarkisian et al.,2006). This association suggests that Filamin A might enhance signaling from Rac1 to JNK, and in doing so may crosstalk with the MT cytoskeleton (see JNK paragraph below).
Although JNK is known to act downstream of Rac to regulate stress response (Johnson and Nakamura,2007), a novel role for JNK in MT regulation critical to radial migration has been reported. JNK expression is strong in the IZ (Hirai et al.,2002; Kawauchi et al.,2003), and its activity is negatively affected by expression of DN Rac1N17 (Kawauchi et al.,2003). Administration of a JNK inhibitor, or in utero electroporation of DN JNK, retards migration (Kawauchi et al.,2003; Hirai et al.,2006), with migrating cells possessing a leading process that is twisted and irregular. Examination of cultured neurons revealed that activated JNK is detected along MTs in processes, and a JNK inhibitor causes twisted, irregular, thick MTs, and shorter neurites, as well as decreased phosphorylation of MT associated protein 1B (Map1B), which is required for MT stability (Kawauchi et al.,2003). Map1B is also present in migrating IZ cells (Cheng etal.,1999), its overexpression overstabilizes MTs and perturbs neuronal migration (Kawauchi et al.,2005), and Map1B knockout mice exhibit abnormal cortical lamination (Gonzalez-Billault etal.,2005). JNK also phosphorylates DCX, which is localized to neurite tips by JNK interacting protein bound to kinesin, to regulate neurite outgrowth and the velocity of migrating neurons (Gdalyahu et al.,2004). The nonphosphorylated forms of Map1B and DCX interact with and stabilize MTs, and phosphorylation of these proteins decreases their affinity for MTs, leading to increased MT dynamics. Thus JNK, through the phosphorylation of these substrates, increases MT dynamics in migrating neurons. Activity of JNK in migrating cortical neurons is regulated by upstream kinases, including the dual leucine zipper kinase (DLK)/MAPK upstream protein kinase/leucine zipper protein kinase and MEKK4/MAP3K4. DLK, like JNK, is predominantly expressed in the IZ (Hirai etal.,2002; Kawauchi et al.,2003). Overexpression of DLK perturbs radial migration (Hirai et al.,2002), and genetic disruption of DLK decreases JNK activity and the phosphorylation of known JNK substrates, including c-Jun and DCX, and impairs axon growth and radial migration of neocortical pyramidal neurons (Hirai et al.,2006). As mentioned above, MEKK4/MAP3K4 deficiency is also associated with migration defects and links JNK with Filamin A (Sarkisian et al.,2006).
Taken together, these findings provide strong support for a role for Rac in radial migration. Perturbation of Rac function, as well as of a number of the Rac-interacting proteins discussed above, generally does not inhibit migration entirely, but rather delays migration. In addition, Rac signaling does not appear to affect cell polarization, but causes defects in the morphology of the leading process. Interestingly, a study on rhombic lip cell migration in the developing chicken cerebellum comes to this same conclusion. In this system, expression of DN RacN17 suppresses the generation of protrusions at the tip of the leading process and impairs leading process extension, but does not alter overall polarity. PI 3-kinase and Pak also regulate leading process formation and morphology, respectively, in these cells (Sakakibara and Horwitz,2006).
Cdc42 in Radial Migration
Although the role of Cdc42 in radial migration has not been as extensively examined compared with Rac, existing studies suggest a role for Cdc42 in this process. In the developing neocortex, perturbation of Cdc42 activity retards radial migration (Konno et al.,2005). In addition, some insight into the role of Cdc42 in CNS migration comes from experiments on Cdc42 in Lis1 signaling (Fig. 7). Lis1 (Pafah1b1) is a subunit of the platelet activating factor acetylhydrolase 1b (Pafah1b) (Hattori et al.,1994), and mutations in LIS1/PAFAH1B1 and associated signaling molecules are associated with lissencephaly. Lissencephaly is characterized by lack of (agyria), or reduction in (pachygyria), normal cerebral surface convolutions, resulting in a “smooth brain,” thickening of the cortex, abnormal cortical lamination, mental retardation, and epilepsy (Reiner et al.,1993; Pang et al.,2008; Spalice et al.,2009). Lis1 is required both for radial migration and for other steps in neuronal development leading up to migration, including maintenance of neuroepithelial stem cells, interkinetic nuclear movement, and progression of neural progenitors through the cell cycle in the VZ, as well as the formation of process outgrowth (Tsai et al.,2005; Yingling et al.,2008).
Regulation of cell motility and migration by Lis1 involves activation of Cdc42 and the formation of a complex that contains Lis1, active GTP-bound Cdc42, IQGAP1, and CLIP-170 (Kholmanskikh et al.,2006). IQGAP1 was identified as an effector of Cdc42 and Rac1 (Hart et al.,1996; Kuroda et al.,1996) and is an actin-binding protein that stabilizes actin filaments at the leading edge (Fukata et al.,1997). It interacts with the MT-stabilizing protein CLIP170 to connect MT plus ends to peripheral actin filaments (Fukata et al.,2002; Watanabe et al.,2004). Activation of Cdc42 and Rac1 appears to target IQGAPs and CLIP-170 to specific cortical areas to polarize MTs and regulate the front-rear polarization of migrating cells (Fukata et al.,2002; Watanabe etal.,2004) and promote axon outgrowth (Wang et al.,2007). In addition to acting as an effector for Cdc42 and Rac1, IQGAP1 inhibits the GTPase activity of Cdc42 and stabilizes the active, GTP-bound form of this GTPase (Ho et al.,1999). In migrating cerebellar granule neurons, calcium influx enhances neuronal motility through Lis1 activation of Cdc42, leading to the perimembrane localization of IQGAP1 and CLIP-170, which presumably tethers MT ends to the actin cytoskeleton to regulate migration. In turn, IQGAP1 may stabilize and thus enhance Cdc42 activity in migrating neurons (Kholmanskikh et al.,2006). Cdc42 and Lis1 also appear to be partners of IQGAP1 in the nonradial migration of neuronal precursor cells of the SVZ and the RMS (Balenci etal.,2007).
Although these findings implicate Cdc42 in radial migration, the contribution of Cdc42 to different stages of the migration cycle, and the various signaling pathways involved, remain to be elucidated. It will be critical to determine where active Cdc42 localizes throughout the migration cycle in CNS neurons, and whether known Cdc42 interactors, such as the conserved Par6 polarity complex, mediate its effects on migration, or whether Cdc42 regulates glial-guided radial migration independent of these proteins.
Rho in Radial Migration
Strict regulation of RhoA levels and activity appear to be required for radial migration. In the developing rodent neocortex, RhoA mRNA expression is high in the premigratory cortical VZ and SVZ, and low in cells migrating in the IZ. In contrast, RhoB mRNA expression is high only in the CP (Olenik et al.,1999; Ge et al.,2006). In the developing cerebellum, RhoA expression is high in cells in the EGL, IGL, and Purkinje cell layer (Richard et al.,2008). Electroporation of the VZ of embryonic mouse cortex with RhoA or DN RhoAN19 shows that ectopic expression of RhoA blocks radial migration, whereas interfering with RhoA function promotes migration (Ge et al., 2006; Nguyen et al., 2006). In addition, loss of a negative regulator of Rho, p190 RhoGAP, causes defects in forebrain development, including impaired layering of the cerebral cortex (Brouns etal.,2000). Taken together, these findings suggest that downregulation of RhoA activity is required for the radial migration of neurons. However, despite these findings, active RhoA has been reported in the leading process of migrating cerebellar GCPs (Guan et al.,2007), suggesting that lower levels of RhoA may still play a role in radial migration (see further below).
The proneural basic helix–loop–helix (bHLH) transcription factors Ngn1 and 2 initiate differentiation and migration in the neocortex by upregulating factors required for migration, such as DCX and p35, and downregulating RhoA. Ectopic expression of Ngn1 down-regulates both RhoA levels and activity, and Ngn2 deficiency increases RhoA expression. Loss of Ngn1, Ngn2, or both, also expands the zone of RhoA expression in the developing neocortex, with higher RhoA expression in the IZ compared with controls (Ge et al.,2006). Importantly, Ngn2 is required for radial migration (Mattar et al.,2004; Schuurmans et al.,2004; Hand et al.,2005; Ge et al.,2006; Nguyen et al.,2006; Heng et al.,2008) and specifies the polarity of the leading process during the initiation of migration by a mechanism that is independent of its transactivation properties and proneural function. The migration defect observed in the Ngn2 knockout mice can be rescued by expressing DN RhoAN19 in cortical progenitors (Hand et al.,2005). Consistent with a negative role for Ngn2 in regulating RhoA, Ngn2 knockout also causes a decrease in expression of two negative Rho GTPase regulators, p190RhoGAP-B/RhoGAP5/ARHGAP5 and srGAP2/formin binding protein 2 (FNBP2) (Mattar et al.,2004; Hand et al.,2005).
RhoA signaling during radial migration is also down-regulated by a signaling pathway that involves Cdk5 and the cyclin-dependent kinase (CDK) inhibitor p27(Kip1) (Fig. 7). Cdk5 phosphorylates and stabilizes p27 to maintain the amount of p27 in postmitotic neurons (Kawauchi etal.,2006). Similar to loss of Cdk5 function (Gilmore et al., 1998), p27 deficiency prevents cortical precursors from migrating to the CP (Kawauchi et al.,2003;2006; Nguyen et al.,2006). Both DN Cdk5 and p27 shRNA expressing cells in the lower IZ are round with thin processes instead of multipolar, but only DN Cdk5 expressing cells in the upper IZ lack a normal leading process (Kawauchi etal.,2006). Interestingly, p27 regulates neuronal differentiation and radial migration by two distinct mechanisms, which are independent of its role in cell cycle regulation. p27 promotes neuronal differentiation by stabilizing Ngn2 protein, an activity carried out by the N-terminal half of the protein, and it promotes neuronal migration by blocking RhoA signaling, an activity that resides in its C-terminal half. Expression of Ngn2 rescues differentiation defects, and expression of DN RhoAN19 rescues the migration defects caused by knock-down of p27 in vivo. Inhibition of RhoA signaling using an inhibitor for the Rho effector Rho-kinase also rescues p27 siRNA migration defects in cortical slices (Nguyen et al.,2006).
By suppressing the RhoA/Rho-kinase pathway, Cdk5-p27 signaling can modulate F-actin in migrating cells. Rho-kinase phosphorylates and activates the LIM (Lin-11, Isl-1, and Mec-3) domain-containing kinases, which in turn phosphorylate and inactivate cofilin, an actin filament depolymerizing/severing factor (Maekawa et al.,1999; Sumi et al.,1999; Ohashi et al.,2000; Ohashi etal.,2000; Amano et al.,2001; Sumi et al.,2001). In fact, p27 decreases cofilin phosphorylation by suppressing the RhoA/Rho-kinase pathway in fibroblasts (Besson et al.,2004), and p27 deficient cortical neurons contain less F-actin than control cells (Kawauchi et al.,2006). A CDK inhibitor, expression of DN Cdk5 and expression of p27 shRNA all cause an increase in cofilin phosphorylation in cultured cortical neurons, and phosphorylation of cofilin induced by the CDK inhibitor can be suppressed by expression of DN Rho-kinase or DN RhoAN19. The importance of cofilin phosphorylation to radial migration is further highlighted by the arrest of neuronal precursors in the IZ of embryonic cortices electroporated with a phosphorylation deficient cofilin mutant (Kawauchi et al.,2006).
Although down-regulation of RhoA levels and activity appear to be required for cortical neuron migration, low levels of RhoA may still be necessary to regulate actomyosin contractility and drive motility. The actin-based motor Myosin II contracts actin filaments to generate the force needed to power cell motility and turn over actin-based adhesions (Gupton et al.,2002; Webb et al.,2004; Gupton and Waterman-Storer,2006; Vicente-Manzanares et al.,2009; Vicente-Manzanares et al.,2009), and Myosin IIB is the predominant Myosin II motor expressed in the nervous system (Kawamoto and Adelstein,1991; Rochlin et al.,1995). Rho-kinase regulates myosin activity downstream of RhoA by directly phosphorylating and activating myosin light chain (MLC), and by phosphorylating and inactivating MLC phosphatase (MLCP), thereby indirectly increasing MLC phosphorylation and activation (Amano et al.,1996; Kimura et al.,1996). Myosin IIB mutant mice display an abnormal pattern of cerebellar foliation and defects in GCP migration along Bergmann glial fibers (Ma et al.,2004). Myosin II motors and F-actin are enriched in the leading process of migrating neurons, and Myosin II activity is required for high actin dynamics in this region, and for centrosomal and somal motility (Schaar and McConnell,2005; Solecki et al.,2009). In cortical neurons, inhibition of Myosin IIB appears to regulate nuclear, but not centrosomal movement (Tsai et al.,2007). The mechanism by which Myosin II regulates centrosomal and somal movement in migrating neurons may involve the conserved polarity protein mPar6α, which localizes to the centrosome and also regulates centrosomal and somal movement (Solecki et al.,2004). Ectopic expression of Par6α inhibits Rho-kinase phosphorylation of MLCP, leading to enhanced MLCP dephosphorylation of MLC, and ultimately a reduction in the actomyosin contractility that drives neuronal migration. Par6 also interacts with MLC and MLC kinase (MLCK), a positive regulator of myosin activity. Disruption of Par6-MLC binding via overexpression of the IQ-like domain of Par6α inhibits MLC phosphorylation and increases the turnover time of F-actin in the leading process of migrating neurons (Solecki et al.,2009) (Fig. 7). In the future, it will be of interest to determine whether low levels of RhoA activity regulate centrosome positioning and nuclear movement during radial glial-guided migration.
Rho/Rho-kinase signaling may also be affected by Filamin A, an actin binding protein associated with the cortical malformation PH (Fox et al.,1998; Feng and Walsh,2004; Sarkisian et al.,2008; Zhou et al.,2010) discussed in detail in the Rac in Radial Migration section above. In addition to binding Rac and Cdc42 signaling components (Ohta et al.,1999; Bellanger et al.,2000; Vadlamudi et al.,2002; Ohta et al.,2006), Filamin A can associate with the Rho GEF Lbc (Pi etal.,2002) and form a complex with Rho-kinase (Ueda et al.,2003), which phosphorylates and stimulates the Rac GAP FilGAP to inactivate Rac (Tseng et al.,2004). Filamin A also promotes the accumulation of p190RhoGAP in lipid rafts (Mammoto et al.,2007). Thus Filamin A appears to be capable of both activating and inactivating the Rho GTPases Rac and Rho (Fig. 7). Filamin A may increase Trio/Rac/Pak signaling during cell protrusion, while decreasing Rho activity through p190RhoGAP. Conversely, during cell retraction, Filamin A may stimulate Lbc to increase Rho/Rho-kinase activity, while decreasing Rac activity through FilGAP (Zhou et al.,2010). However, despite all of these possibilities, how Filamin A interacts with Rho GTPase signaling during radial migration remains to be defined.
A recent study suggests that positive regulation of RhoA by Semaphorin-Plexin signaling may facilitate radial migration. Semaphorin 3A (Sema 3A) expression is highest in superficial cortical layers (Polleux et al.,2000), and Sema 3A and 3F have been shown to set the directionality of cortical neuron migration. Knockdown of Sema3A receptors, including Neuropilin and Plexin subunits, as well as the application of exogenous Sema3A, delays radial migration (Chen et al.,2008). Mice lacking Plexin-B2 exhibit neural tube closure defects and a variety of other developmental brain defects, including exencephaly, enlargement of the ventricles, hypotrophy of the VZ, disruption of the ventricular wall, and ventricular ectopias. Plexin-B2 is required for proliferation of VZ neuroblasts, and proliferation and migration of GCPs in the cerebellum, dentate gyrus, and olfactory bulb. In a heterologous system, Sema 4C binding to Plexin-B2 activates the receptor tyrosine kinase ErbB-2 and RhoA (Fig. 7), suggesting that these molecules may play a role in proliferation and the ability of neural precursors to migrate directionally (Deng etal.,2007).
There is also a possibility that Rho signaling may regulate termination of radial neuronal migration through a link with the heterotrimeric G proteins Gα12 and Gα13 and the orphan G protein-coupled receptor GPR56 (Fig. 7). The heterotrimeric G proteins Gα12 and Gα13 link G-protein-coupled receptors to actomyosin-based cellular contractility and are required for the proper termination of radial migration by cortical neurons. Simultaneous ablation of both Gα12 and Gα13 genes results in neuronal ectopia of the cerebral and cerebellar cortices due to overmigration of cortical plate neurons and cerebellar Purkinje cells, respectively (Moers et al.,2008). Gα12 and Gα13 couple with the orphan G protein-coupled receptor GPR56, which is highly expressed in neural progenitor cells and inhibits their migration. Coupling of GPR56 with Gα12 and Gα13 induces Rho-dependent activation of serum-responsive element and NFkappaB transcription and actin reorganization, which can be inhibited by expression of the RGS domain of the p115 Rho-specific GEF (p115 RhoGEF RGS) and DN RhoAN19. In addition, the effects of an anti-GPR56 antibody that acts as an agonist and inhibits migration can be attenuated by p115 RhoGEF RGS, C3 exoenzyme that inhibits Rho, and GPR56 knockdown. Although the GPR56-related migration experiments were performed using neurospheres, and therefore were not in vivo, these findings provide a potential mechanism whereby Rho might participate in the termination of migration during cortical development (Iguchi et al.,2008). Indeed, although RhoA is mainly expressed in the VZ at early embryonic stages of neocortical development, it appears also to be present in the CP at later stages after cells have finished migrating (Olenik et al.,1999). RhoA might therefore transduce stop signals that prevent neurons from migrating too far.
Taken together, these studies suggest that a reduction in RhoA levels and activity are required to commence migration, and persistently low levels may regulate actin reorganization, actomyosin contractility, and transcription required for the mechanics of motility and the cessation of movement.