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Over the past decade, remarkable progress has been made in our understanding of the mechanisms of cell migration. In general, cell migration involves polarization of the cell in the direction of movement, the formation of a broad lamellipodium and filopodia, and directed vesicle trafficking toward the leading edge (Ridley et al.,2003; Heasman and Ridley,2008). Just behind the leading edge, Myosin II motors act in concert with integrin-based adhesions to provide traction for cell locomotion (Gupton et al.,2002; Ridley et al.,2003; Giannone etal.,2004; Hu et al.,2007; Vicente-Manzanares et al.,2009). Adhesions are disassembled at the rear of the cell to allow detachment, and spike-like, actin-driven filopodia serve as sensors to explore the local environment (Ridley et al.,2003; Heasman and Ridley,2008). Although many neuronal migrations in the developing brain can be described by this general model, several highly specialized modes of movement establish the neuronal layers during corticogenesis. These include tangential migrations along emerging axon tracts, chain migrations of neurons ensheathed in a glial network, and neuronal migrations along the radial glial fiber network (Fig. 1).
The Rho family GTPases are critical regulators of key steps in cell migration. They are low-molecular weight guanine nucleotide binding proteins, which in general function as binary molecular switches that cycle between an active GTP-bound and inactive GDP-bound state. Their activity can be positively influenced by guanine nucleotide exchange factors (GEFs) that promote the exchange of GDP for GTP (Schmidt and Hall,2002; Rossman et al.,2005), and negatively influenced by GTPase activating proteins (GAPs) that increase the intrinsic rate of GTP hydrolysis of the GTPase (Bernards,2003; Bernards and Settleman,2004) and by guanine nucleotide dissociation inhibitors that sequester and solubilize the GDP-bound GTPase and prevent the exchange of GDP for GTP (Zalcman etal.,1999; Hoffman et al.,2000; DerMardirossian and Bokoch,2005). Active, GTP-bound Rho GTPases transduce upstream signals by interacting with effector molecules (Van Aelst and D'Souza-Schorey,1997; Bishop and Hall,2000; Gundersen,2002; Bokoch,2003; Riento and Ridley,2003; Burridge and Wennerberg,2004; Govek et al.,2005; Bustelo etal.,2007; Kurisu and Takenawa,2009). Spatial and temporal specificity of Rho GTPase signaling are achieved in part via spatiotemporal regulation of regulatory molecules (Etienne-Manneville and Hall,2002), GEFs that link Rho GTPases to specific effectors through scaffolding molecules (Buchsbaum et al.,2002, 2003; Jaffe etal.,2004), post-translational modifications such as lipid modification and phosphorylation (Ward et al.,2004; Roberts etal.,2008), and by ubiquitin-mediated proteosome degradation (Senadheera et al.,2001; Doye et al.,2002; Wang et al.,2003; Ward et al.,2004; Riento etal.,2005; Visvikis et al.,2008). In general during migration, Cdc42 plays an integral role in polarizing the centrosome and defining the direction of movement, and Rac and Cdc42 are classic regulators of actin-based lamellipodia and filopodia formation, respectively, and Rho regulates cell contractility. All three of these GTPases also play a role in cell adhesion (Ridley,2001; Fukata etal.,2003; Raftopoulou and Hall,2004). Although the effects of the Rho GTPases on non-neuronal cell migration have been studied extensively, the part that they play in CNS migrations is only beginning to be elucidated with the advent of advanced techniques that allow us to examine the contribution of individual molecules in migrating neurons in vivo.
This review will provide a brief description of migrations critical for cortical and cerebellar histogenesis, and highlight the role of the Rho GTPases, their regulators, and effectors in these different modes of CNS migrations.
CELL MIGRATIONS IN CORTICAL DEVELOPMENT
Interkinetic Nuclear Movements in the Cortical Ventricular Zone
In the mouse, neural precursors in the neuroepithelium lining the third ventricle of the emerging telencephalic vesicle begin to proliferate just after neural tube closure (E9.25). Like all epithelia, the neuroepithelium of the telencephalic ventricular zone (VZ) has an apical-basal polarity. In contrast to most epithelia in the mouse embryo, however, the apical surface of progenitor cells in the telencephalic neuroepithelium is oriented toward the ventricular surface, where adherens junctions are dependent on the conserved Par6 polarity complex. During neurogenesis, interphase progenitor cells span the epithelium, moving toward the ventricular surface just before mitosis. During G1, the nucleus of proliferating progenitor cells moves away from the ventricular surface as the progenitor cell extends toward the pial surface. During the S phase of the cycle, the nucleus is located close to the basal, pial surface, and moves back toward the apical, ventricular surface just before entering mitosis (Sauer and Walker,1959) (Fig. 2). The translocation of the nucleus throughout the cell cycle is termed interkinetic nuclear migration. Cyclic interkinetic nuclear migrations involve dynamic changes in the microtubule (MT) and actin cytoskeletons, as well as cytoskeletal and adhesion regulatory molecules (Tsai et al.,2005; Xie et al.,2007; Del Bene et al.,2008; Latasa et al.,2009; Norden et al.,2009; Schenk et al.,2009; Zhang etal.,2009; Buchman et al.,2010; Ge et al.,2010; Sottocornola et al.,2010). The Rho GTPase family members, Rac1 and Cdc42, are among the proteins that regulate neurogenesis and interkinetic nuclear movements.
Recent studies have revealed a role for Rac1 in the earliest stages of neural progenitor production and fate specification. Rac1 is present at the ventricular surface during cortical development (Minobe et al.,2009), and Rac1 deficiency in the telencephalic VZ of mouse embryos causes a reduction in neural progenitors, in part due to increased apoptosis, resulting in microcephaly (Chen et al.,2009). Rac1 inhibition, using a dominant negative (DN) mutant, also retards nuclear migration and causes failures in cytokinesis (Minobe et al.,2009). These findings suggest that Rac1 may regulate self-renewal, survival, and interkinetic nuclear migration of neocortical progenitor cells.
Cdc42 deficiency in telencephalic progenitor cells also causes defects in forebrain development, characterized by an increase in neuron number, defective radial glia/progenitor fibers, and holoprosencephaly. Cdc42 protein is enriched at the apical/ventricular side of the neuroepithelium and is present in basally located postmitotic neurons. Loss of Cdc42 abolishes the localization of Par complex proteins, E-cadherin, β-catenin, and Numb to adherens junctions at the apical surface of neural progenitors, causing a loss of adherens junctions, failure of interkinetic nuclear migration, and an increase in mitosis at the basal surface (Cappello et al.,2006; Chen et al.,2006). Apical localization of Cdc42 and other polarity proteins depends on the myristoylated alanine-rich C-kinasesubstrate protein, which is required for radial progenitor localization at the VZ and formation of the radial glial scaffold (Weimer et al.,2009). The formation of adherens junctions and the interaction of Cdc42 with polarity proteins also depends on ceramide in the apical membrane of the neuroepithelium (Wang et al.,2008). Taken together, these findings suggest that Cdc42 regulates adherens junction formation, interkinetic nuclear migration, and self-renewal of VZ progenitors, as well as radial glial scaffold formation.
Insight into how Cdc42 affects the fate of VZ progenitors comes from experiments on the Par polarity complex members, Par6, Par3, and atypical Protein Kinase C (aPKC), in neocortical development. The binding of active, GTP-bound Cdc42 (or Rac1) to Par6 (Joberty etal.,2000; Lin et al.,2000; Qiu et al.,2000; Noda et al.,2001; Garrard et al.,2003) activates aPKC to phosphorylate proteins (Qiu et al.,2000; Etienne-Manneville and Hall,2001). A loss of aPKCλ also affects adherens junction formation by causing apical process retraction and impairs interkinetic nuclear migration, resulting in disordered neuroepithelial tissue architecture (Imai et al.,2006). Loss of both Cdc42 and Par3 causes a decrease in self-renewing progenitors and an increase in neurons, whereas overexpression of Par3 and Par6 promotes the generation of self-renewing progenitors. Similarities in the effects of Par3 and Par6 on progenitor self-renewal in vitro and in vivo suggests that the consequent defects in progenitor self-renewal resulted from a reduction in Par protein levels, as opposed to a loss of tissue polarity or coordinated apicobasal fate determinants (Costa et al.,2008). Thus, Cdc42 promotes self-renewing progenitor cell divisions at the expense of neurogenic differentiation in the developing cerebral cortex by regulating the levels and/or activity of apically localized Par-complex proteins.
Development of Secondary Germinal Zones
Beginning on about day E13 in the mouse, a secondary germinal zone, called the subventricular zone (SVZ), forms above the VZ and expands significantly as cortical development proceeds (Molyneaux et al.,2007). Neurogenesis in this zone produces a variety of neurons, including precursors of olfactory bulb GABAergic neurons, which migrate from the cortical SVZ into the olfactory bulb along a pathway called the rostral migratory stream (RMS) (Luskin,1993). Proliferating precursors in the RMS migrate at speeds up to 122 μm/h (Wichterle et al.,1997; Murase and Horwitz,2002) as a chain of neurons ensheathed by a protective layer of glial cells in a posterior to anterior direction to populate the olfactory bulb (Lois et al.,1996). This migration continues in the adult brain, where recent studies show that newly generated neurons produce Slit1, a diffusible protein whose receptor, Robo, is produced by the astrocytic meshwork through which these neurons migrate (Doetsch et al.,1997; Kaneko et al.,2010). This finding suggests that neuron–glial interactions control glial morphological differentiation needed to maintain the RMS glial network. Repulsion of migratory cells from the anterior SVZ by Slit involves down-regulation of Cdc42 activity via the Slit-Robo GAP (srGAP) 1 (Wong et al.,2001). In addition to providing a thoroughfare for RMS migration, astrocytes in this region also concentrate BDNF released by vascular endothelial cells (Snapyan et al.,2009), take up GABA released by migrating neurons (Bolteus and Bordey,2004), and secrete other factors that promote migration. Several different adhesion receptor systems are necessary for RMS migration, including polysialiated NCAM (Battista and Rutishauser,2010) and α1, αv, and β1 integrins (Murase and Horwitz,2002). Deletion of Rac1 in the telencephalic VZ using Fox1-Cre severely impairs the tangential migration of ventral telencephalon-derived olfactory and cortical interneurons. This failure of tangential migration may be due to a failure in acquisition of migratory competency during the differentiation of interneuron progenitors, rather than a general defect in motility, and may be dependent, at least in part, on limited extension of corticofugal axons (Chen et al., 2007). Although Rac1 is required for interneurons to gain competency to migrate, during migration Dlx transcription factors repress the Rac/Cdc42 effector p21-activated serine/threonine kinase (Pak)3 to restrain neuritic growth and facilitate tangential migration (Cobos et al., 2007).
Neuronal Migrations during Preplate and Cortical Plate Stages of Cortical Development
In the mouse, the earliest neurons generated in the telencephalic VZ become postmitotic on, or before, the 11th day of gestation (E11). These early-born, polymorphic neurons settle in a narrow laminar zone above the neuroepithelium, the preplate (Caviness,1982; Wood,1992; McConnell et al.,1994; Del Rio et al.,2000; Zecevic and Rakic,2001) (Fig. 3). They include Cajal–Retzius cells, GABAergic interneurons, and future subplate projection neurons (Caviness,1982; Marin-Padilla,1998; Del Rio et al.,2000). Preplate neurons migrate within the thin preplate zone in a dorsoventral direction at an average speed of 18 μm/h. During periods of movement, preplate neurons rapidly extend and retract a broad lamellipodium (Schneider et al., 2011).
Between E11 and E12, preplate neurons pioneer the first axon projections of the neocortex, with some preplate neurons extending descending axons to subcortical targets and others extending corticocortical axons to targets in the ipsilateral or contralateral hemisphere via the anterior commissure. Like tangentially migrating preplate neurons, the growth cones of extending axons also exhibit a general mode of motility that includes the formation of a broad lamellipodium and leading edge. In addition, axon guidance receptor systems localized to the tips of actin-based filopodia control the directionality of movement.
The Rho GTPases are key mediators of signaling by guidance cues in neurons. RhoA signaling increases Myosin II activity to promote axon retraction and growth cone collapse, whereas Rac1 and Cdc42 regulate lamellipodial and filopodial formation in growth cones and promote axon extension and guidance (Gallo and Letourneau,2004). A recent report shows that Rac1-induced lamellipodia formation in growth cones requires the Wiskott–Aldrich syndrome protein family verprolin-homologous protein (WAVE) complex and Arp2/3, which regulates actin nucleation and polymerization to induce cytoskeletal remodeling (Tahirovic et al.,2010). Of note, pharmacological inhibition of Rho/Rho kinase signaling in precerebellar neurons enhances axon extension and impairs migration toward a netrin-1 source, whereas pharmacological inhibition of Rac and Cdc42 impairs neurite outgrowth without affecting migration. This suggests that axon outgrowth and nuclear translocation rely on distinct signaling pathways (Causeret et al.,2004). The role of Rho GTPases in axon formation and guidance has been reviewed in (Govek et al.,2005; Koh,2006; Yoshimura et al.,2006; Witte and Bradke,2008; Yi et al.,2010).
Tangential migrations, first described by McConnell and colleagues, occur in the intermediate zone (IZ) along pioneering axons (O'Rourke etal.,1992; Anderson et al.,1997). These tangentially migrating cells include massive populations of interneurons that emigrate into the telencephalon from the ganglionic eminence in the basal forebrain. Interneurons migrate into the cortex through the marginal zone and the IZ beginning at E14-16 in the mouse, with huge numbers moving into the cortex as the neuronal laminae form. Tangentially migrating interneurons undergo a specialized form of migration that involves cyclical steps of leading process extension or branching followed by nuclear translocation. Interestingly, leading process extension and branching ceases during nuclear translocation. A swelling also forms that contains the centrosome and the Golgi apparatus, accompanied by centriole splitting, and moves into the leading process before nuclear movement. Nuclear movement is dependent on the activity of Myosin II, which accumulates in the rear of the cell (Bellion et al.,2005). A particularly unique and interesting aspect of interneuron migration is the extension or branching of a new leading process along the pre-existing leading process or on the cell body when the neuron changes direction (Polleux et al.,2002; Martini et al.,2009).
Formation of the Cortical Plate and Neuronal Layers
In the developing neocortex, neurons generated after E13 form a second transient layer, the cortical plate, in the middle of the preplate. During cortical plate formation, preplate neurons segregate into a superficial marginal zone containing Cajal–Retzius cells and a deeper layer, the subplate (Caviness,1982; Wood,1992; McConnell et al.,1994; Del Rio et al.,2000; Molnar et al.,2006) (Fig. 3). Just before the cortical plate forms, a series of changes occur in the morphology and orientation of preplate neurons, leading to a pseudocolumnar patterning of preplate neurons. At E12.5, a stage before preplate splitting, preplate neurons have a polymorphic shape that is randomly aligned relative to the radial axis. Over the next 12 h, preplate neurons undergo changes in polarity from a polymorphic shape to a bipolar morphology, and assemble into a transient, columnar pattern as the preplate segregates into the superficial marginal zone and underlying subplate (Schneider et al., 2011) (Fig. 4). Our recent observations of active changes in preplate/subplate neuron morphology and orientation during preplate splitting suggest that polarity signaling pathways, as well as Rho GTPase pathways, might be required for the radially migrating neurons to migrate past subplate neurons during formation of the cortical plate.
Glial-Guided Neuronal Migration and the Formation of Neuronal Laminae
After the segregation of preplate cells into the marginal zone (Kostovic,1974; Luskin and Shatz,1985), cortical plate and subplate neuronal precursors undergo a mode of migration unique in cortical regions of the developing mammalian brain. Precursors of cortical large output neurons generated in the VZ of the developing neocortex adhere to and migrate on the fibers of radial glial cells that span the entire cortical wall, with an apical end-foot at the ventricular surface and a basal process at the pial surface. This gliophilic mode of migration was first proposed by Rakic from Golgi and EM studies (Rakic,1972). As development proceeds, later born cohorts of neuronal precursors migrate past existing cortical layers to form an inside-out sequence of six cortical layers (Rakic,1974; Luskin and Shatz,1985). In the developing human neocortex, directed migrations establish the principal neuronal layers by the end of the second trimester (Sidman and Rakic,1973). In addition to providing a substrate for neuronal migrations, radial glia serve as neural progenitors (Noctor et al.,2001, 2002). Genetic analyses by Anthony and Heintz show that (1) radial glia are neuronal progenitors in all regions of the developing CNS, (2) the vast majority of neurons in all brain regions derive from radial glia, and (3) radial glia within different regions of the CNS pass through their neurogenic stage of development at distinct time points (Anthony et al.,2004). Thus, radial glia serve as a scaffold for neuronal progeny to migrate and populate the cortical plate.
The Developing Cerebellum
The cerebellum, like the neocortex, consists of an outer cortical structure and set of subcortical nuclei, the cerebellar nuclei, which project axons to cerebellar targets. Progenitors in the neuropeithelial ventricular zone (VZ) along the dorsomedial surface of the IVth ventricle give rise to the principal output neuron of the cerebellar cortex, the Purkinje cell, neurons of the cerebellar nuclei and more than half a dozen types of cerebellar interneurons, including Golgi, basket, and stellate cells. A secondary germinal zone forms along the anterior portion of the rhombic lip, which generates the cerebellar granule neuron, as well as a subpopulation of neurons of the cerebellar nuclei (Wang et al.,2005; Fink et al.,2006) and neurons of several precerebellar nuclei (Dymecki and Tomasiewicz,1998; Wingate and Hatten,1999; Machold and Fishell,2005). Direct evidence for precursor cell migration along radial glia in the embryonic cerebellum comes from confocal imaging studies on transgenic mice expressing markers for transcription factors in neurons of the cerebellar nuclei and the Purkinje neurons (Morales and Hatten, 2006). Between E10 and E12, postmitotic precursors of neurons of the cerebellar nuclei, identified by their expression of the transcription factors Lhx2/9, Meis 1/2, and Irx3, migrate radially along the emerging glial fiber system to form a superficial zone across the dorsal surface of the cerebellar anlagen. Between E11 and E14, postmitotic precursors of the Purkinje neuron, identified by expression of the LIM transcription factors Lhx1 and Lhx5, migrate away from the VZ along radial glial fibers and assemble into a broad zone in the core of the anlagen (Morales and Hatten,2006).
As discussed above, progenitors in the secondary germinal zone of the cerebellar anlagen, the EGL, give rise to cerebellar granule neurons. The remarkable abundance of granule cell progenitors (GCPs), up to several million in a neonatal mouse, has facilitated correlated in vitro and in vivo studies on their glial-guided neuronal migration. As GCPs exit the cell cycle, they move to deeper aspects of the EGL where they assume a bipolar shape and extend two axons, called parallel fibers. Localization of the centrosome plays a key role in determining where a neurite or process will form. During parallel fiber formation, the centrosome localizes to one pole of the cell where a neurite extends parallel to the axis of the EGL. Subsequently, the centrosome moves to the opposite pole of the cell and extends a second neurite parallel to the axis of the EGL (Zmuda and Rivas,1998). GCPs then extend a descending, leading process perpendicular to the axis of the EGL, attach to the radial fiber of a Bergmann glial cell, and migrate through the molecular layer, past the Purkinje cell layer, to the inner granular layer (IGL), trailing a T-shaped axon behind (Rakic,1971; Edmondson and Hatten,1987; Komuro and Rakic,1995, 1998). After migration into the emerging IGL, multiple dendrites form, which establish synaptic connections with in-growing afferent mossy fibers (Fig. 5). The peak period of GCP proliferation and granule neuron migration occurs around postnatal day (P)8.
Cellular and Molecular Mechanisms of Radial Glial-Guided Migration
As first described by Rakic, glial-guided neuronal migration involves alignment of postmitotic neurons with a system of radial glial fibers during periods of cortical formation in the neocortex, hippocampal formation, and cerebellum (Rakic,1971;1972;1974). Interestingly, live imaging studies on migrating neurons from these three brain regions (Edmondson and Hatten,1987; Gasser and Hatten,1990b), as well as on migrating neurons from heterotypic brain regions (Gasser and Hatten,1990a), revealed that neuronal migration is not dependent on region-specific features of radial glia. Indeed, a similar mode of movement occurs when CNS neurons migrate on glass fibers with the same geometric parameters as glial fibers (Fishman and Hatten,1993). Live imaging studies of cerebellar granule neuron migration in vitro revealed features of glial-guided migration that were distinct from the general crawling movement of metazoan cells. These unique features include the extension of a highly polarized, leading process in the direction of migration, the assembly of an interstitial adhesion junction beneath the cell soma (Gregory et al.,1988), localization of actomyosin contractile motors in the proximal portion of the leading process (Solecki et al.,2009), and the formation of a perinuclear cage of tubulin to maintain the posterior positioning of thenucleus. The migration cycle involves forward movement of the centrosome into the proximal portion of the leading process preceding translocation of the neuronal nucleus (Edmondson and Hatten,1987; Solecki et al.,2004), the activation of actomyosin motors located in the proximal aspect of the leading process (Solecki et al.,2009), and the release of the adhesion junction, allowing the cell soma to glide forward along the glial fiber until a new adhesion forms (Fig. 6).
Recent, live imaging studies demonstrate the contribution of actin and MT regulatory molecules in this process. Dynamic rearrangement of the actin cytoskeleton occurs in the proximal region of the leading process, where Myosin II motors localize. Myosin II activity, which is regulated by the conserved mPar6α polarity complex, is required for both centrosomal and somal motility (Solecki et al.,2004, 2009). The MT cytoskeleton is further regulated by cytoplasmic dynein and its cofactors LIS1 and Doublecortin (DCX) to couple centrosomal and nuclear movement (Faulkner et al.,2000; Smith et al.,2000; Coquelle etal.,2002; Tai et al.,2002; Tanaka et al.,2004a; Tsai et al.,2005; Mesngon et al.,2006; Tsai etal.,2007; Umeshima et al.,2007). Given the key role of the Rho GTPases in regulating the actin and MT cytoskeletons, the role of the Rho GTPases in radial migration will be discussed in detail below.
Insight into the critical role of adhesion and receptor trafficking during neuronal migration comes from studies on receptor systems that function in glial-guided neuronal migration, including Astrotactin (ASTN1), Neuregulin/ErbB4, and BDNF. The neuronal protein ASTN1 (Edmondson et al.,1988; Fishell and Hatten,1991) is expressed by neurons in both the cerebellum and cerebral cortex (Zheng et al.,1996). Mice lacking ASTN1 have slowed cerebellar granule neuron migration, a decrease in neuron–glial binding and abnormal Purkinje cell development compared with wild-type (WT) littermates (Adams et al.,2002). Recent studies have identified a second member of the Astrotactin receptor family, ASTN2, which regulates the cell surface expression and trafficking of ASTN1 critical for granule cell migration (Wilson et al.,2010). The importance of trafficking to neuronal migration is further highlighted by the finding that inhibition of clathrin-mediated endocytosis brings migration to a halt (Zhou et al.,2007; Wilson et al.,2010).
Neuregulin binds ErbB4 on the glial surface and provides signals that maintain glial process formation (Anton et al.,1997; Rio et al.,1997), and BDNF stimulates granule neuron migration (Borghesani etal.,2002). Surprisingly, integrin-based adhesions, which are essential for most types of cell migration, are not required for glial-guided neuronal migration in the developing brain (Edmondson et al.,1988; Fishell and Hatten,1991; Belvindrah etal.,2007).
Insight into the importance of receptor trafficking to neuronal migration also comes from experiments on the reeler mutant mouse. In the reeler mouse, preplate segregation into a superficial marginal zone and deeper subplate zone fails, and the arrival of cortical plate stage neurons generates a broad “superplate” rather than a cortical plate (Caviness,1982; Goffinet,1984; Tissir and Goffinet,2003). Although the failure of preplate splitting has been attributed to defects in the cessation of the migration of later-generated neurons destined for the cortical plate (Caviness,1982; Goffinet,1984), recent studies on changes in polarity and orientation of preplate neurons show that these neurons in rl/rl neocortex fail to adopt a polarized shape and align with the radial axis like their counterparts in WT neocortex. These studies identify the earliest known defect in reeler cortical patterning and suggest that defects in preplate neuron polarity precede defects in neuronal migration during early stages of reeler corticogenesis. The failure of preplate neurons to transiently polarize in reeler mice further suggests that the protein and pathway responsible, Reelin (RELN), might converge on conserved polarity signaling pathways to control neuronal alignment and polarity during preplate splitting.
The most widely accepted model for RELN signaling postulates that RELN, an extracellular matrix glycoprotein, binds to low-density lipoprotein (LDL)receptors, including apoliprotein E receptor 2 (apoER2) and very LDL (VLDL) receptors (D'Arcangelo etal.,1999; Hiesberger et al.,1999; Trommsdorff et al.,1999; Hack et al.,2007), and activates Src family tyrosine kinases, which subsequently phosphorylate vertebrate homologs of the Drosophila protein Disabled 1 (DAB1) (Howell et al.,1997; D'Arcangelo et al.,1999; Hiesberger et al.,1999; Howell et al.,1999; Jossin etal.,2003; Kuo et al.,2005). Experiments by Mikoshiba and coworkers have suggested a novel role for DAB1 in RELN signaling, namely that DAB1 regulates the expression of cell surface RELN and trafficking of RELN receptors (Morimura et al.,2005). This model is based on the observation that RELN is internalized by clathrin-dependent endocytosis, and the fact that adaptor proteins with a PTB domain, including DAB1, regulate intracellular trafficking of transmembrane receptors that contain the cytoplasmic NPXY motif (Mishra et al.,2002). In this model, expression of DAB1 increases the expression of RELN receptors on the cell surface and tyrosine phosphorylation of DAB1 initiates intracellular trafficking of both RELN and its receptors (Morimura et al.,2005), which affects cortical plate formation and cortical lamination.
The ability of the Rho GTPases to regulate cell adhesion and motility (Ridley,2001; Fukata et al.,2003; Raftopoulou and Hall,2004), as well as clathrin-dependent and independent endocytosis (Symons and Rusk,2003), suggests that they may regulate the formation and dissolution of adhesion junctions through receptor trafficking during neuronal migration.
The Rho GTPases in RADIAL GLIAL-GUIDED NEURONAL MIGRATION
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).
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.
Rnd2 in Radial Migration
The Rnd proteins constitute a unique branch of the Rho GTPases that have low intrinsic GTPase activity and are thought to be CA. As a result, they are also insensitive to the effects of classical Rho GTPase regulators, including GEFs and GAPs. There are three Rnd family members, called Rnd1, Rnd2, and Rnd3/RhoE. The Rnd proteins have been shown to regulate neurite formation, axon extension, and dendritic development, as well as cell-cycle control (Riento et al.,2005; Chardin,2006). Rnd1 and Rnd3/RhoE proteins regulate reorganization of the actin cytoskeleton by reducing cellular levels of GTP-bound RhoA through a p190RhoGAP mechanism, and RhoE sequesters Rho-kinase to prevent it from phosphorylating MLCP (Riento et al.,2003; Wennerberg et al.,2003; Riento et al.,2005; Chardin,2006). In contrast, Rnd2 interacts with the effector Pragmin to stimulate RhoA/Rho-kinase activity and induce cell contraction (Tanaka et al.,2006). Rnd2 also interacts with the protein Rapostlin in a GTP-dependent manner, which binds directly to MTs and regulates reorganization of both actin filaments and MTs to induce neurite branching (Fujita et al.,2002). Importantly, Rnd2 has been shown to play a critical role in cortical radial migration (Nakamura et al.,2006; Heng et al.,2008).
During cortical development, Rnd2 expression is intense in basal/intermediate progenitors and radially migrating neurons in the SVZ and IZ, respectively, weak in the VZ progenitors, and sharply downregulated in cells in the CP (Nakamura et al.,2006; Heng et al.,2008). In utero electroporation of Rnd2 or CA Rnd2V16 resulted in a failure of neurons to migrate to upper cortical layers. However, cells electroporated with DN Rnd2N21 migrated similar to control cells, a result which likely reflects an inability of this mutant to utilize and titrate out GEFs the way a traditional DN Rho GTPase mutant does (Nakamura et al.,2006). Knockdown of Rnd2 by ex vivo electroporation of cortices with Rnd2 siRNAs, followed by organotypic slice cultures, markedly inhibited radial migration without affecting the proliferation of cortical progenitors, their specification to a cortical neuron identity, or the organization of the radial glial scaffold. Neurons with compromised Rnd2 expression also exhibited morphological defects, including an increased fraction of IZ neurons with a multipolar morphology and increased length of the longest neurite, and those with a unipolar or bipolar shape had branched leading processes compared with control neurons. Neuronal processes in Rnd2 knockdown cells were also more unstable. Not only did Ngn2 directly induce Rnd2 expression, and Rnd2 knockdown defects in radial migration were similar to those observed upon deletion of the Ngn2 gene, but also Rnd2 expression in Ngn2 deficient neurons resulted in a remarkable rescue of their ability to migrate (Heng et al.,2008). Thus, Rnd2 is a major effector of Ngn2 function in the promotion of migration.
Over the last decade, remarkable progress has been made in understanding the mechanisms of neuronal migration. The advent of in utero electroporation, advanced live imaging techniques, and the increasing availability of conditional mutant mouse strains have allowed us to examine the contribution of individual molecules to specific stages of neuronal development. Using these experimental methods, we have learned that the Rho GTPases play critical and unique roles in neurogenesis and radial migration. During neurogenesis in the cortex, Rac and Cdc42 regulate the self-renewal of neural progenitors, and, Cdc42, in particular, regulates interkinetic nuclear migration. During radial migration, Cdc42 likely polarizes migrating neurons, whereas Rac regulates leading process formation and protrusion. Although down-regulation of RhoA is required for migration, low levels likely persist to regulate actomyosin contractility required to generate the force needed to drive motility. In addition, Rnd2 has emerged as a critical and strong regulator of radial migration. These Rho GTPases act within multiple signaling pathways, including those associated with cortical malformations, which ultimately converge on the actin and MT cytoskeletons. Thus, the Rho GTPases integrate and propagate signals from various upstream regulators to produce the morphological changes needed to progress through development and undergo directed movement. As the function of the Rho GTPases during neurogenesis and migration is dependent on strict regulation of not only their levels but also their activity, it will be interesting to see how Rho GTPase levels and activity are localized and regulated in different subcellular compartments of live, developing and migrating neurons in vivo. It will also be critical to define how all of these signaling pathways required for radial migration come together to produce the complex architectonics of the human brain associated with higher cognitive functions.
We are grateful to Mr. James Duffy for help with the illustrations in Figures 3, 6, and 7, to Dr. Carol A. Mason at the College of Physicians and Surgeons of Columbia University for providing the drawing in Figure 5, and to Mr. William Carey for proofreading the manuscript. Supported by STARR (M.E.H. and L.V.A.) and National Institutes of Health—National Institute of Neurological Disorders and Stroke Grant RO1NS 051778-05 (M.E.H.) and National Institute of Mental Health Grant RO1MH082808 (L.V.A.).