In addition to apico-basal polarity, another hallmark of NEs retained by RGs is interkinetic nuclear migration (INM), a process whereby nuclei change position along the apico–basal axis during the course of the cell cycle. In NEs, this INM spans the entire apical–basal axis of the cell, with the nucleus migrating to the basal side during the G1 phase of the cell cycle, staying at the basal side during S phase, migrating back to the apical side during the G2 phase and undergoing mitosis at the ventricular surface. In RGs, the same mitotic behavior occurs, except that is confined to the portion of the cell in the VZ (**Figure 4**, ❷). As a consequence of this movement, the neuroepithelium and the VZ appear pseudo-stratified (Taverna and Huttner, 2010). The precise role of INM during cortical neurogenesis is still an unresolved question. INM might allow packing an increasing number of progenitor cells within a limited ventricular surface or it might regulate progenitor fate by influencing the exposure of progenitor nuclei to proliferative vs. neurogenic signals (Taverna and Huttner, 2010; Spear and Erickson, 2012).

Both microtubule-based motors and actomyosin seem to participate in either direction of INM, although to a different extent depending on the system (Taverna and Huttner, 2010; Lee and Norden, 2013). In the developing cerebral cortex, a few studies have implicated the Rho GTPases in the regulation of this process.

**FIGURE 4 | Development of projection neurons in the mouse cerebral cortex.** The neural stem/progenitor cells of the cerebral cortex or radial glial cells (RGs) are highly polarized cells that are attached to one another in the ventricular zone (VZ) by apically located adherens junctions (AJ) ❶. Their nuclei migrate during cell cycle progression from a basal position during S phase to an apical position during mitosis (M), and the nuclei of the daughter cells migrate back to enter S phase on the basal side of the VZ, in a process called interkinetic nuclear migration (INM) ❷. During the peak of neurogenesis, most radial glial cells divide asymmetrically with a vertical cleavage plane ❸. In these divisions, one daughter remains a RG and continues to divide at the ventricular surface, whereas the other detaches from the ventricular surface, move radially away to the subventricular zone (SVZ)/lower intermediate zone (IZ) and acquires a multipolar shape ❹. Then, nascent neurons become bipolar, extending a leading process toward the pial surface and a trailing process in the opposite direction ❺. Upon multi to

In particular Cdc42, Rac1, and Rnd3 have been shown to control the basal-to-apical movement. Indeed, this movement is delayed in *Cdc42* deficient mice (Cappello et al., 2006) or after electroporation of a dominant negative (DN) form of Rac1 (Minobe et al., 2009) (**Table 1**). However, it is not known whether microtubule or actomyosin networks mediate the effects of Rac1 and Cdc42. In contrast, the impairment of basal to apical INM after *Rnd3* silencing in the embryonic cerebral cortex is rescued by coexpression of a constitutively active form of cofilin (cofilinS3A), demonstrating that Rnd3-mediated disassembly of actin filaments coordinates the cellular behavior of RGs during INM at least during the apical nuclear movement (Pacary et al., 2013).

#### **Rho GTPases AND REGULATION OF PROGENITOR CELL DIVISION, PROLIFERATION AND CELL FATE**

At early stages of corticogenesis, NEs divide symmetrically to self-renew and expand their pool. Following the transition to the bipolar transition, newborn neurons establish contacts with RG fibers and subsequently use them as a scaffold to migrate to the upper part of the cortical plate (CP) using a mode of migration called locomotion ❻. During this phase the trailing process becomes the axon and extends to its final destination. Once cortical neurons reach the upper part of the CP and right after their leading process makes contact with the marginal zone (MZ), they detach from the RG fibers and execute a terminal somal translocation ❼. The leading process then gives rise to the apical dendrite, which initiates local branching in the MZ ❽. Basal dendrites subsequently appear as well as oblique side branches emerging from the apical shaft ❾. At this stage, the cell body of early-born neurons translocate ventrally as neurons born at later stages bypass their predecessors. The final step in cortical projection development is the apparition and maturation of spines. For example, in layer V pyramidal neurons, spines are morphologically mature at P21 on apical dendrites ❿.

RG fate, some progenitor cells begin to divide asymmetrically to generate neurons directly or indirectly through the production of IPs or oRGs (Laguesse et al., 2014).

After their generation by asymmetric division of RGs, IPs, also called basal progenitors, retract their apical and basal processes, exhibit a multipolar morphology and migrate basally (**Figure 4**, ❸ and ❹) before they undergo mitosis. This second pool of proliferative progenitors undergo one or more symmetric cell divisions (Noctor et al., 2004), which significantly increases the yield of cortical neurons derived from a single RG. The accumulation of these dividing progenitors in basal regions starts around E13 and it determines the formation of the subventricular zone (SVZ). Whereas NEs and RGs express identical markers like the transcription factor Pax6 (Gotz et al., 1998), IPs are identified by the absence of Pax6 and by the expression of the transcription factor Tbr2 (Englund et al., 2005).

#### **Table 1 | Regulation of cortical projection neuron development by Rho GTPases (***in vivo* **studies).**

#### **Table 1 | Continued**

*VZ: ventricular zone, SVZ: subventricular zone, IZ: intermediate zone, CP: cortical plate, INM: interkinetic nuclear migration, CA: constitutively active, DN: dominantnegative.*

oRGs, also known as basal radial glia cells, were first discovered in human and ferret brains (Fietz et al., 2010; Hansen et al., 2010), and were initially proposed to be a specific feature of gyrencephalic brains. However, they have also been identified in the rodent brain, where they account for less than 10% of total cortical progenitors vs. 40% in human (Hansen et al., 2010; Wang et al., 2011), and in lissencephalic primate brains (Garcia-Moreno et al., 2012; Kelava et al., 2012). oRGs arise from the division of RGs as they delaminate from the apical surface and translocate their nuclei in the outer portion of the SVZ, where they start dividing. To note, this translocation of the soma along the basal fiber toward the CP, a process termed mitotic somal translocation, requires activation of the Rho effector ROCK (Ostrem et al., 2014). In contrast to IPs, oRGs maintain molecular characteristics of RGs such as expression of Pax6 and can divide either symmetrically to expand their number or asymmetrically to self-renew and give birth to new neurons (Hansen et al., 2010; Reillo et al., 2011).

The balance between proliferation and differentiation of these different categories of progenitors is tightly regulated and is fundamental for the generation of appropriate number of cortical neurons. Among the Rho GTPases, RhoA, Rac1, RhoG, and Rnd3 have a crucial role in the control of this balance. Indeed, conditional deletion of *RhoA* in cortical progenitors using *FoxG1Cre* mice causes hyperproliferation, which results in the expansion of the progenitor pool and exencephaly-like protrusions (Katayama et al., 2011). Similarly, the loss of *RhoA* by *Emx1-Cre* mediated recombination increases proliferation in a region-specific manner within the cerebral cortex, starting at occipital regions at E14 and later at E16 in rostral parts, and this phenotype is associated with an aberrant location of Pax6+ and Tbr2+ progenitors (Cappello et al., 2012) (**Table 1**). In contrast to RhoA, the forebrain-specific loss of *Rac1* by *FoxG1Cre* leads to a SVZ-specific reduction in proliferation, a concomitant increase in cell cycle exit and premature differentiation (Chen et al., 2009; Leone et al., 2010) (**Table 1**). How RhoA and Rac1 differently affect proliferation of cortical progenitors is not known. However, studies in other cellular systems have shown that RhoA and Rac1 influence the levels of cyclins during G1 progression. Interestingly, Rac1, but not Rho, stimulate cyclinD1 transcription when ectopically expressed in cells (Jaffe and Hall, 2005). In addition, RhoA might regulate proliferation of cortical progenitors during cytokinesis through its action on F-actin and myosin II into the actomyosin contractile ring (Marzesco et al., 2009) or through its action on actomyosin filaments at the cell cortex which influence mitotic spindles and the plane of cell division (see next paragraph) (Jaffe and Hall, 2005). Rnd3 has also been shown to control specifically the proliferation of basal progenitors via cyclinD1 but in an opposite manner, i.e., *Rnd3* silencing increases SVZ proliferation (Pacary et al., 2013). Finally, RhoG, another Rac-related Rho GTPase expressed in the VZ/SVZ (**Figure 3**), also promotes neural progenitor cell proliferation in the mouse cerebral cortex (**Table 1**) through phosphatidylinositol 3-kinase (PI3K) signaling (Fujimoto et al., 2009).

In the developing cerebral cortex, cleavage plane orientation remains predominantly vertical (planar division) during the period of symmetrical division prior to neurogenesis, and throughout the period of asymmetrical division during neurogenesis (**Figure 4**, ❸) (Morin and Bellaiche, 2011). In these asymmetric divisions, one daughter remains a RG and continues to divide at the ventricular surface, whereas the other loses its apical attachment and becomes an IP. Fewer RGs undergo oblique or horizontal divisions, and these divisions have been proposed to generate oRGs (Morin and Bellaiche, 2011; Shitamukai and Matsuzaki, 2012).

In the mouse cerebral cortex, Rnd3 is required to maintain the vertical orientation of the cleavage plane during RG divisions. Indeed, when *Rnd3* is knockdown the fraction of RGs dividing with an oblique or horizontal cleavage plane is increased (Pacary et al., 2013). Interestingly *Rnd3*-silenced cells prematurely leave the VZ, enter the SVZ while transiently maintaining their radial glial molecular phenotype, thus showing similarities with oRGs (Pacary et al., 2013). In addition, co-electroporation of cofilinS3A restores vertical cleavage-plane orientation in *Rnd3* silenced progenitors, thus indicating that Rnd3 maintains the vertical orientation of apical divisions by remodeling the actin cytoskeleton (Pacary et al., 2013). RhoA might be also important to determine the orientation of cortical progenitor divisions as suggested by a study in the chick neuroepithelium, in which the expression of a DN form of RhoA results in random spindle orientation (Roszko et al., 2006). Inversely, deletion of *Cdc42* does not influence spindle orientation in the developing cerebral cortex (Cappello et al., 2006). However, as mentioned previously, the loss of *Cdc42* causes defects in apical process maintenance, thereby leading to an increased number of progenitors dividing at basal rather than apical positions. These progenitors convert to an SVZ fate as shown by the increase of Tbr2+ progenitors and a decrease of Pax6+ population in the mutants, which ultimately leads to a higher rate of neuron generation (Cappello et al., 2006).

#### **Rho GTPases AND REGULATION OF RADIAL MIGRATION**

After detachment from the ventricular surface in the VZ, nascent neurons move radially away to the SVZ/lower intermediate zone (IZ), where they acquire a multipolar shape (**Figure 4**, ❹). During this phase, multipolar neurons actively extend and retract dynamic processes and tend to migrate tangentially in an apparent random fashion (Noctor et al., 2004; Jossin and Cooper, 2011). Then, neurons become bipolar, extending a leading process toward the pial surface and a trailing process in the opposite direction (nascent axon) (**Figure 4**, ❺). Upon multi to bipolar transition, neurons establish dynamic contacts with RG fibers and subsequently use them as a scaffold to migrate to the upper part of the CP using a mode of migration called locomotion (**Figure 4**, ❻). This movement is characterized by repetitive cycles of synchronized steps. First, a cytoplasmic dilation forms in the proximal region of the leading process. Second, the centrosome moves toward the swelling and finally the nucleus translocates toward the centrosome, a process known as nucleokinesis. This migration cycle then starts again confering a saltatory advancement to the locomoting neurons. Finally, once cortical neurons have reached the uppermost area of the CP and right after their leading process makes contact with the MZ, they detach from the RG fibers and execute a terminal somal translocation to settle in their appropriate final position (**Figure 4**, ❼) (Nadarajah et al., 2001). Rac1, Rnd2, and Rnd3 are three Rho GTPases with specific functions in the control of the migratory process in the developing cerebral cortex: Rac1 signaling regulates leading process formation (Kawauchi et al., 2003; Konno et al., 2005), Rnd2 is critical for the multi to bipolar transition (Heng et al., 2008; Pacary et al., 2011) and Rnd3 is important for nuclear-centrosome coupling during locomotion (Pacary et al., 2011).

Conditional knockout of *Rac1* in the forebrain, *in utero* electroporation of DN or constitutively active (CA) forms of Rac1 as well as *Rac1* shRNA or wild-type Rac1 have demonstrated a requirement for this Rho GTPase in radial migration (Kawauchi et al., 2003; Konno et al., 2005; Chen et al., 2007; Kassai et al., 2008; Yang et al., 2012a). *Rac1* deletion using the *FoxG1Cre* (Chen et al., 2007) or *Emx1Cre* line (Kassai et al., 2008) disturbs radial migration, but the defects observed in these mice are less severe than those observed after electroporation of the DN form of Rac1 (N17-Rac1). While the migration of nascent neurons seems to be only delayed in the mutants, the inhibition of Rac1 activity with N17-Rac1 leads to an accumulation of electroporated cells in the IZ (Kawauchi et al., 2003; Konno et al., 2005; Yang et al., 2012a). In addition, N17-Rac1 expressing cells in this cortical domain fail to extend a leading process and are instead round, with short and irregular processes (Kawauchi et al., 2003). Interestingly, the electroporation of *CA-Rac1* (*V12Rac1*) induces a similar phenotype (Konno et al., 2005; Yang et al., 2012a) indicating that cycles of Rac1 activation and inactivation and thus a fine regulation of Rac1 activity is important for proper morphological polarization and migration. The difference obtained between conditional knockout mice and the electroporation of dominant mutant might reflect the ability of this mutant as well as of CA mutant to interfere with the activity of other Rho GTPases, possibly through the competitive binding with regulators like RhoGDIs (Boulter et al., 2010). Nevertheless, the requirement of Rac1 for proper radial migration has been further confirmed recently with *Rac1* shRNA (Yang et al., 2012a). Indeed, the silencing of *Rac1* blocks radial migration and disrupts the formation of the proximal cytoplasmic dilation in the leading process of migratory cortical neurons (Yang et al., 2012a). Interestingly, in this study, the authors also show that electroporation of wild-type *Rac1* promotes neuronal migration and that POSH, a Rac1-interacting scaffold protein, recruits activated Rac1 to the plasma membrane. At this site, activated Rac1 regulates actin remodeling and controls the dilation of the leading process, two key events that promote centrosomal movement and soma translocation (Yang et al., 2012a). To note, another report provides the evidence that migration defects caused by loss of *Rac1* in *Foxg1Cre* mice may be due, at least in part, to defects in radial glial organization (Leone et al., 2010).

*In vivo* loss of function studies on the atypical Rho GTPase Rnd3 revealed that its knockdown in migrating neurons results in enlarged leading processes with numerous branches and increases centrosome-nucleus distance in the CP, indicative of disrupted nuclear-centrosome coupling during locomotion (Pacary et al., 2011). In contrast, *Rnd2*-deficient neurons fail to leave the IZ and display long processes at the multipolar stage, suggesting that Rnd2 is critical for the multipolar to bipolar transition that occurs in the IZ (Heng et al., 2008; Pacary et al., 2011). In addition, the two proteins fail to compensate for each other during neuronal migration, further indicating that they play distinct roles in this process (Pacary et al., 2011). Despite these different functions, both Rnd2 and Rnd3 regulate neuronal migration by inhibiting RhoA. Indeed, FRET analysis *in vivo* showed that *Rnd2* or *Rnd3* silencing increases RhoA activity in cortical cells and *RhoA* knockdown rescues the migratory defects associated with *Rnd2* or *Rnd3* loss of function. The inhibitory effect of Rnd3 on RhoA activity depends on its interactions with p190RhoGAP, whereas Rnd2's RhoA inhibitory activity does not. Further, although both Rnd2 and Rnd3 regulate actin dynamics in migrating neurons, only Rnd3 promotes neuronal migration by inhibiting RhoAmediated actin polymerization and remodeling (Pacary et al., 2011). Interestingly, the distinct subcellular localization of Rnd2 and Rnd3 and the resultant modulation of RhoA activity in different cell compartments underlie the difference in their effects. Rnd3 owes its distinct role in neuronal migration to its localization and interaction with RhoA at the plasma membrane. Rnd2 is expressed in early endosomes and can replace Rnd3 in migrating neurons if it is targeted to the plasma membrane by replacement of its carboxyl-terminal domain with that of Rnd3 (Pacary et al., 2011). However, the mechanisms by which Rnd2 promotes neuronal migration and inhibits RhoA remains unknown. The finding that Rnd3 and Rnd2 control different phases of radial migration by inhibiting RhoA in different cell compartments suggests that in cortical neurons, RhoA acts dynamically in different cellular domains to control different aspects of the migratory process.

The functions of the other most studied members of the Rho GTPase family, RhoA and Cdc42, in the control of radial migration are less well-understood. When *DN-Cdc42* (*N17Cdc42*) or *CA-Cdc42* (*V12Cdc42*) are electroporated *in utero*, radial migration is inhibited but this effect is not as strong as that seen with *DN-Rac1* or *CA-Rac1* (Konno et al., 2005). In addition, the role played by Cdc42 in migrating neurons might be different from that of Rac1 since the former is mainly localized to the perinuclear region on the side of the leading process whereas the latter is expressed at the plasma membrane (Konno et al., 2005). Similarly, strict regulation of RhoA levels and activity appear to be required for radial migration *in vivo* (Nguyen et al., 2006; Pacary et al., 2011; Cappello et al., 2012; Azzarelli et al., 2014; Tang et al., 2014). The general view proposes that RhoA activity must be downregulated to promote radial migration of pyramidal neurons. However, the analysis of *RhoA* knockout using *Emx1-Cre* suggests that RhoA is dispensable for radial migration. In this mutant, the deletion of *RhoA* generates migration defects that are only secondary to radial glia scaffold disruption. Indeed, when *RhoA* knockout cells are transplanted in a wild type environment, they migrate normally, suggesting that there is no cell-autonomous requirement for RhoA activity during radial migration (Cappello et al., 2012). How to reconcile the data showing the requirement of RhoA for radial migration with the fact that *RhoA*-depleted neurons normally migrate in a wild type environment is still an open question. One possibility is that compensatory mechanisms might occur to replace RhoA function in *RhoA* conditional knockout cortices. Indeed, it has been shown that the related GTPase RhoB is strongly up-regulated in the absence of RhoA (Ho et al., 2008). RhoB and RhoC might substitute for RhoA activity during cortical neuron migration. These compensatory mechanisms may be in operation only when *RhoA* expression is completely abrogated by knockout deletion.

#### **Rho GTPases AND REGULATION OF NEURONAL POLARIZATION**

Cortical neurons exist in a number of different shapes and sizes, although a mature neuron typically has several dendrites that receive inputs from presynaptic neurons and one axon that relays information to post-synaptic neurons. The formation of axondendrite polarity is thus crucial for a neuron to establish the precise information flow within the brain. Axons and dendrites differ in morphology, function, and protein and organelle composition. Although the development of these processes has been studied extensively *in vitro* using cultured embryonic hippocampal neurons, the corresponding developmental processes *in vivo* are still unclear. During corticogenesis, early electron microscopic studies revealed that projection neurons initiate their axons during migration whereas significant dendrite growth occurs after the cells have reached their final position (Shoukimas and Hinds, 1978).

#### *Rho GTPases and regulation of axon formation, growth, guidance and branching*

Time-lapse imaging revealed that multipolar cells in the IZ, after extending and retracting their short processes for several hours, suddenly elongate a long process tangentially. These cells then transformed into a bipolar shape, extending a pia-directed leading process (future apical dendrite), and migrate radially leaving the tangential process behind, forming an "L-shaped" axon (**Figure 4**, ❻) (Hatanaka and Yamauchi, 2013). Thus, during migration, the trailing process becomes the axon and extends while being guided to its final destination. Interestingly, a recent study has shown that the interaction between multipolar cells and the preexisting axons of early-born neurons is critical for axon specification. Indeed, once one of the neurites of a multipolar cell contacts the pioneering axons from the early-born cortical neurons, this neurite is stabilized, becomes the axon and extends rapidly (Namba et al., 2014). The duration of axon elongation is however variable according to the targeted area, which is more or less distant according to the final layer position of the cortical neuron (Molyneaux et al., 2007). For example, axons of corticofugal neurons in layer V reach the spinal cord around postnatal day P7 in mouse. Finally, upon reaching its target area, extensive axonal branching occurs during the formation of presynaptic contacts with specific post-synaptic partners (during the second and third postnatal week in the mouse cortex) (Lewis et al., 2013).

The regulation of axon development by Rho GTPases has been mainly studied *in vitro* and reviewed elsewhere (Govek et al., 2005; Hall and Lalli, 2010). However, their specific roles *in vivo* are less well-understood. Cdc42 seems however to be clearly required during cerebral cortex development for the efficient establishment of axonal polarity and growth. Indeed, cortices of *Cdc42* conditional knockout mice crossed with *Nestin-Cre* mice exhibit a widespread reduction of axonal tracts (Garvalov et al., 2007). This phenotype is accompanied by a specific increase in the phosphorylation (inactivation) of the Cdc42 effector cofilin (Garvalov et al., 2007). The axonal defects in *Cdc42* knockouts might be due to the increased levels of inactive cofilin since the deletion of this actin depolymerizing protein results in polarity defects analogous to the ones seen after *Cdc42* ablation (Flynn et al., 2012). In agreement with these data, the growth and organization of callosal axonal fiber tracts are also disrupted in *Cdc42* deficient mice obtained after mating *Cdc42* floxed mice with *hGFAP-Cre* line (Yokota et al., 2010). In addition to Cdc42, a role for Rnd2 in cortical axon extension has been suggested in a study showing that COUP-TFI, a transcription factor crucial for corticogenesis and arealization, promotes callosal axon elongation by finely regulating *Rnd2* expression levels (Alfano et al., 2011).

In contrast to Cdc42, the loss of *Rac1*, using *Foxg1–Cre* mice, does not prevent axonal outgrowth in cortical neurons (Chen et al., 2007). However, in these mutants, the anterior commissure is absent, and the axons of the corpus callosum and the hippocampal commissure fail to cross the midline. A similar phenotype is observed in *Rac1/Emx1Cre* knockout mutants (Kassai et al., 2008), demonstrating that Rac1 controls axon guidance rather than neuritogenesis. In addition, the thalamocortical and corticothalamic axons show defasciculation or projection defects in *Rac1/FoxG1–Cre* mutants (Chen et al., 2007), whereas corticospinal and corticothalamic projections are not affected in *Rac1/Emx1Cre* mice (Kassai et al., 2008). This phenotypic discrepancy might be due to the different pattern of Cre expression in these two Rac1 mutants. Indeed, in *FoxG1–Cre* mice, the recombinase is expressed in other regions than the telencephalon such as the thalamus (Hebert and McConnell, 2000) which might affect the development of thalamocortical projections. To note, Rac3, the other member of the Rac subfamily expressed in the brain, does not seem to have redundant functions with Rac1 since *Rac3* knockouts do not show any obvious developmental defects in the cortex (Corbetta et al., 2005).

The *in vivo* role of RhoA in cortical axon development has not been thoroughly examined but the cortical axons in *RhoA/Emx1– Cre* knockout mutants show correct morphology and projections (Cappello et al., 2012). Nevertheless, RhoA might act as a mediator for activity-dependent branch formation as suggested by a study performed in cortical explants (Ohnami et al., 2008).

#### *RhoGTPases and dendrite/spine formation*

In contrast to the axon, dendrites in cortical neurons form after migration ends. These structures are highly branched and this feature gives them the appearance of a tree. Initially, all excitatory cortical neurons exhibit the common shape of "pyramid," which is characterized by a prominent apical dendrite. This first dendrite derives from the leading process and branches out in an apical tuft that terminates in layer I (**Figure 4**, ❽). With time, basal dendrites appear as well as oblique side branches emerging from the apical shaft (**Figure 4**, ❾) (Whitford et al., 2002). The dendritic trees and consequently the overall neuronal shapes vary greatly within the cortex, with neurons of layers II, III, V, and VI acquiring a pyramidal morphology, whereas those of layer IV predominantly having non-pyramidal morphologies. Therefore, even if the initial stages of dendrite formation are very similar, further maturation determines the final neuronal morphology. For example, spiny stellate neurons in layer IV start out with a pyramidal morphology, but then acquire a stellate morphology, by retracting their apical dendrite at an early postnatal age (Vercelli et al., 1992).

The final step in the acquisition of a mature dendritic morphology is the development of spines (**Figure 4**, ❿). These small dendritic protrusions, which contain receptors and other proteins necessary for synaptic transmission, begin to appear in the first postnatal week in mice, when the arborization of the apical and basal dendrites becomes more complex. When pyramidal neurons reach their mature morphology, they have a highly complex dendritic arbor and are covered with spines. The timing of spine development is variable among neurons that occupy different layers or cortical areas (Huttenlocher and Dabholkar, 1997; Whitford et al., 2002).

The role of Rho GTPases in dendrite and spine formation has been mainly addressed in culture. By manipulating Rho, Rac1, and Cdc42 activities in a number of experimental systems, it has become clear that each of these Rho GTPases plays a prominent role in the development of dendrite structure and that interplay between them determines the complexity of the dendritic tree (Newey et al., 2005). Studies are generally consistent with a key role for RhoA in controlling dendritic length and for Rac and Cdc42 in regulating dendrite branching and remodeling. More precisely, RhoA activation has a negative effect on dendritic arbor growth, whereas Rac1 and Cdc42 activation promote this process. Similarly, RhoA inhibits, whereas Rac1 and Cdc42 promote spine formation and maintenance (Newey et al., 2005). However, considering the large body of *in vitro* work, it is surprising how little is known about their roles *in vivo* in the context of cortical development. To our knowledge, only one study has addressed this role directly *in vivo*. Rosario and colleagues have shown that *in utero* electroporation of a CA form of Cdc42 under the control of the post-mitotic promoter NeuroD1 decreases dendrite branching and complexity in layer II/III pyramidal neurons at postnatal stages (Rosario et al., 2012). The other evidences are indirect and come from *in vivo* studies showing involvement of Rho GTPase regulators, namely GAPs and GEFs, in this process. These regulators include GAPs, such as NOMA-GAP, srGAP2 (also called FNBP2), Myo9b and RICS, as well as GEFs like kalirin (see Section Upstream Regulators of Rho GTPases and Cortical Projection Neuron Development and **Table 2**).

#### **Rho GTPases AND REGULATION OF CORTICAL NEURON DEATH/SURVIVAL**

Programmed neuronal cell death, or apoptosis, is essential for proper cerebral cortex development, resulting in the refinement of nascent neuronal innervation and network formation (Nikolic et al., 2013). In the mouse, neuronal apoptosis takes place in the first 30 postnatal days, with a peak at P5, mostly pronounced in cortical layers II–IV. This wave of apoptosis accounts for a loss of approximately 30% of neuronal content in the cerebral cortex from birth to adulthood (Heumann et al., 1978; Heumann and Leuba, 1983).

Among the Rho GTPases, RhoA is of particular interest with respect to regulation of postnatal apoptosis in the cerebral cortex. Indeed, by engineering a mouse line in which a dominantnegative RhoA mutant (N19–RhoA) is specifically expressed in neurons, Sanno and colleagues have demonstrated that the inhibition of RhoA activity reduces the amount of apoptosis occurring in the postnatal cortex and results in a concomitant increase in the density and absolute number of neurons in the adult cortex (Sanno et al., 2010). Interestingly, the change in neuronal density in the N19–RhoA cortex is attributable to an increase in the number of excitatory projection neurons and not in that of the interneuron population, which originates in the ventral telencephalon (Sanno et al., 2010).

Besides the well-established programmed cell death naturally occurring in the postnatal brain, more recent studies indicate the existence of an earlier wave of programmed cell death affecting neural progenitors and nascent neurons (Yeo and Gautier, 2004). This early wave of cell death appears to play an even more critical role in determining the final size of the brain (de la Rosa and de Pablo, 2000; Kuan et al., 2000). In mouse, this death occurs between E12 and E16 within the VZ and IZ of the cerebral cortex (Blaschke et al., 1996, 1998; Thomaidou et al., 1997). Interestingly, the forebrain specific deletion of *Rac1* mediated by *FoxG1Cre* enhances apoptosis in VZ and SVZ progenitors, mainly around E14.5 (Chen et al., 2009; Leone et al., 2010); this effect partially contributes to a decrease in neural progenitors observed during mid-to-late telencephalic development (Chen et al., 2009). While Rac1 is required for survival of both VZ and SVZ progenitors, Cdc42 plays a dispensable role in cell survival during corticogenesis, as indicated by a comparable number of apoptotic cells in the cortex of control mice and *Cdc42/Nestin–Cre* knockout mutants (Peng et al., 2013).

#### **UPSTREAM REGULATORS OF Rho GTPases AND CORTICAL PROJECTION NEURON DEVELOPMENT**

A number of *in vivo* studies showing involvement of GEFs and GAPs in cortical projection neuron development further support a critical role of the Rho GTPase family in this process and are summarized in **Table 2**. Studying the functions of GAPs and GEFs not only provides an indirect way to clarify the roles played by Rho GTPases in specific steps of corticogenesis, but it also contributes to the overall understanding of the entire pathways activated downstream of Rho proteins. For example, the loss of *NOMA-GAP*, a Cdc42-specific GAP, leads to an oversimplification of cortical dendritic arborization, as well as an hyperactivation of Cdc42. Remarkably, these dendritic defects can be partially restored by genetic reduction of post-mitotic *Cdc42* levels, demonstrating that the post-mitotic inhibition of Cdc42, mediated by NOMA-GAP, is a necessary requirement for dendritic branching during cortical development (Rosario et al., 2012). In this study, the authors further show that *in utero* expression of active cofilin is sufficient to restore postnatal dendritic complexity in *NOMA-GAP*-deficient animals. Therefore, these data support a model, whereby, during cortical dendritic development, cofilin activation is positively regulated by NOMA-GAP through the inhibition of Cdc42 (Rosario et al., 2012). Interestingly, only the dendritic complexity of layer II/III neurons is affected by the genetic ablation of *NOMA-GAP*, whereas layer V pyramidal neurons develop normal dendrites. How NOMA-GAP regulates dendrite development only in specific layers, although this GAP is expressed in all cortical layers (Rosario et al., 2012), is still an open question. It is possible that other Cdc42-GAPs, which

#### **Table 2 | Regulation of cortical projection neuron development by GAPs and GEFs (***in vivo* **studies).**

**Table 2 | Continued**

are selectively expressed in the unaffected layers, might substitute for the loss of *NOMA-GAP* function. Alternatively, some neurons might be more sensitive to NOMA-GAP activity, because they exhibit higher active Cdc42 levels, which could be the result of layer-specific environmental signals or differentially expressed Cdc42-GEFs.

One difficulty with these studies is to determine whether the phenotype observed is due to the inhibition or activation of a specific Rho GTPase or of several members, since these regulators usually show activity against multiple Rho GTPases. For instance, RICS has been classified in **Table 2** as a Rho GAP, because the defects on dendrites induced by *RICS* knockdown are rescued by an inhibition of RhoA signaling (Long et al., 2013) and might thus be due to a modulation of Rho activity. However, it has been shown that RICS prefers Cdc42 over Rac1 or RhoA as a substrate (Simo and Cooper, 2012). Also, it is important to remember that some of the phenotypes described in **Table 2** might not be related to the GAP or GEF activity itself, but other protein domains might be involved. For example, α2-chimaerin, a Rac-GAP, has been shown to regulate radial migration, not through its GAP activity, which is instead dispensable, but through the association with the microtubule-associated protein CRMP-2 (Ip et al., 2012). Similarly, DOCK7 is a Rac-GEF that controls INM independently of its GEF activity (Yang et al., 2012b). The functions of GAPs and GEFs that are independent from GTPase activation or GDP-GTP exchange, respectively, have not been included in **Table 2**.

#### **Rho GTPases AND CEREBRAL CORTEX MALFORMATIONS**

As described previously, the development of the cerebral cortex is remarkably complex and tightly organized. Disruption of any of the overlapping steps that contribute to this process can result in profound and stereotypical cortical malformations. In view of the multiple regulatory functions played by Rho GTPases during cerebral cortex development, it is thus not surprising that their forebrain specific suppression leads to cortical malformations. Accordingly, Cappello et al. found that *Emx1-Cre* mediated deletion of *RhoA* causes three types of malformations in the mouse cerebral cortex (Cappello et al., 2012; Cappello, 2013). First, the adult mutant cerebral cortex is about 1.3-fold thicker than control. This megalencephaly phenotype has been linked to the increased proliferation observed in the *RhoA* conditional mutant (see Section Rho GTPases and Regulation of Progenitor Cell Division, Proliferation and Cell Fate and **Table 1**). The second malformation observed in these mutants is a subcortical band heterotopia, which is characterized by a heterotopic cortex formed of ectopic neurons embedded within the white matter and underlying a normotopic cortex. Interestingly, the formation of this double cortex may not result from direct defects in migrating neurons, but rather from defective radial glia fibers that neurons use as a scaffold to migrate. Indeed, as mentioned previously (see Section Rho GTPases and Regulation of Radial Migration and **Table 1**), *RhoA* null neurons migrate normally when transplanted into wild-type cerebral cortex, whereas the converse is not the case, probably because of the strongly disorganized RG processes in the mutant cortices (Cappello et al., 2012). The last cortical abnormality observed in *Emx1-Cre RhoA* null mutants is the formation of cobblestones or neuronal ectopias at the basal side of the developing cerebral cortex. These neuronal protrusions beyond layer I may result from the increase speed of migrating nascent neurons and/or from the aberrant RG endfeets (Cappello, 2013). Interestingly, *FoxG1Cre RhoA-*deficient embryos also exhibit expansion of the neural progenitor pool and exencephaly-like protrusions (Katayama et al., 2011).

In contrast to *RhoA* deletion, which induces excessive proliferation, *Rac1* loss of function in the mouse cerebral cortex reduces progenitor cell proliferation and increases apoptosis, which may be the major causes of microcephaly observed in the mutants (Chen et al., 2009; Leone et al., 2010). The suppression of *Cdc42* in the cortex also affects the overall cortical morphology. Indeed, *Cdc42*-deficient telencephalon fails to bulge or separate into two cerebral hemispheres, resulting in holoprosencephaly (Chen et al., 2006). This phenotype may result from the essential role of Cdc42 in establishing the apico-basal polarity of RGs and subsequently of the telencephalic neuroepithelium, which is needed for the expansion and bifurcation of cerebral hemispheres (Chen et al., 2006).

Interestingly, *NOMA-GAP* deficiency also leads to a decrease of cortical thickness in different cortical areas (Rosario et al., 2012). This reduction was observed in adult mice, but also at early postnatal stages, suggesting that cortical thinning is due to a defective developmental process. Since the absence of *NOMA-GAP* does not impact the early stages of cortical development, including neuronal birth, survival, fate determination and migration, cortical thinning in these mutants may arise from defective formation of cortical dendritic trees (Rosario et al., 2012).

#### **CONCLUDING REMARKS**

Altogether the above studies highlight the crucial roles played by the Rho GTPase family in the regulation of cerebral cortex development and emphasize that a better understanding of these functions might help to clarify the etiology of several cortical malformations. Further work is required to fully characterize the contribution of the different Rho GTPases expressed in the developing cerebral cortex as well as the downstream signaling involved and the mechanisms regulating their expression and/or activity. Another major challenge for the future will be to understand how the signals through the different Rho GTPases as well as the other small GTPases of the Ras superfamily (Ras, Rab, Ran, Arf) are integrated in nascent cortical neurons and how they are spatially and temporally controlled during cortical development.

Abnormal signaling through Rho GTPases is associated with cognitive dysfunction (Newey et al., 2005; De Filippis et al., 2014) and recent findings have showed their involvement in the development and progression of neurodegenerative diseases (Antoine-Bertrand et al., 2011). Studies on Rho GTPase functions *in vivo* thus represent an underexplored territory that may hold therapeutic potential.

#### **ACKNOWLEDGMENTS**

This work was supported by INSERM and Marie Curie Actions (Intra European Fellowship). Roberta Azzarelli is supported by a Medical Research Council (MRC) post-doctoral fellowship and Thomas Kerloch is recipient of a studentship from the Ministère de l'Enseignement Supérieur et de la Recherche.

#### **REFERENCES**

Yoshizawa, M., Kawauchi, T., Sone, M., Nishimura, Y. V., Terao, M., Chihama, K., et al. (2005). Involvement of a Rac activator,P-Rex1, in neurotrophinderived signaling and neuronal migration. *J. Neurosci.* 25, 4406–4419. doi: 10.1523/JNEUROSCI.4955-04.2005

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 22 October 2014; accepted: 11 December 2014; published online: 07 January 2015.*

*Citation: Azzarelli R, Kerloch T and Pacary E (2015) Regulation of cerebral cortex development by Rho GTPases: insights from in vivo studies. Front. Cell. Neurosci. 8:445. doi: 10.3389/fncel.2014.00445*

*This article was submitted to the journal Frontiers in Cellular Neuroscience.*

*Copyright © 2015 Azzarelli, Kerloch and Pacary. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Polarity transitions during neurogenesis and germinal zone exit in the developing central nervous system

# **Shalini Singh and David J. Solecki \***

Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, TN, USA

#### **Edited by:**

Takeshi Kawauchi, Keio University School of Medicine/PRESTO, JST, Japan

#### **Reviewed by:**

Orly Reiner, Weizmann Institute of Science, Israel Yasuhiro Itoh, Harvard University, USA

#### **\*Correspondence:**

David J. Solecki, Department of Developmental Neurobiology D2025C, St. Jude Children's Research Hospital, 262 Danny Thomas Place-MS 323, Memphis, TN 38105, USA e-mail<EMAIL_ADDRESS>
During neural development, billions of neurons differentiate, polarize, migrate and form synapses in a precisely choreographed sequence. These precise developmental events are accompanied by discreet transitions in cellular polarity. While radial glial neural stem cells are highly polarized, transiently amplifying neural progenitors are less polarized after delaminating from their parental stem cell. Moreover, preceding their radial migration to a final laminar position neural progenitors re-adopt a polarized morphology before they embarking on their journey along a glial guide to the destination where they will fully mature. In this review, we will compare and contrast the key polarity transitions of cells derived from a neuroepithelium to the well-characterized polarity transitions that occur in true epithelia. We will highlight recent advances in the field that shows that neuronal progenitor delamination from germinal zone (GZ) niche shares similarities to an epithelialmesenchymal transition. Moreover, studies in the cerebellum suggest the acquisition of radial migration and polarity in transiently amplifying neural progenitors share similarities to mesenchymal-epithelial transitions. Where applicable, we will compare and contrast the precise molecular mechanisms used by epithelial cells and neuronal progenitors to control plasticity in cell polarity during their distinct developmental programs.

**Keywords: neurogenesis, neuronal progenitor, neuroepithelial, neuronal polarity, cell junction, epithelial mesenchymal transition, delamination**

#### **INTRODUCTION**

The developing central nervous system, including the spinal cord, retina and brain, occupies a complex developmental landscape wherein neural stem cells are born and then proliferate, differentiate and migrate long distances to form intricate networks, all of which ensure proper CNS functioning. Notably, almost all of the diverse cell types comprising the CNS originate from the neuroepithelium lining the embryonic neural tube and neural plate. Self-renewing multipotent cells (neural stem cells) orient themselves along the apical-basal axis in a single layer, conferring a highly polarized structure on this germinal niche. Subsequent symmetric and asymmetric division of the neural stem cells imparts a pseudostratified appearance to neuroepithelium, in which the nuclei undergo interkinetic nuclear migration while the apical-basal surfaces of the cells remain anchored through intercellular junctions (**Figure 1A**; Haubensak et al., 2004; Götz and Huttner, 2005). Alteration of polarity signaling cascade or cell adhesion dynamics leading to improper neural development substantiates the architectural organization of the neuroepithelium (Ayala et al., 2007; Métin et al., 2008; Roussel and Hatten, 2011). As development proceeds these progenitors must commit to a specific neuronal fate and migrate to their final destinations. This step requires them to sever ties with the ventricular zone (VZ), undergo a transition in polarity, change their adhesive preference and delaminate. Our understanding of key components and signaling

cascades, such as the Par polarity complex and its interplay with adhesion molecules such as cadherins, nectins, claudins and junctional adhesion molecules (JAMs), has advanced considerably (Tsukita and Furuse, 1999; Mizoguchi et al., 2002; Costa et al., 2008; Ishiuchi et al., 2009; Famulski et al., 2010), but some key questions remain, including: What are the specific biological processes that precede delamination? What initiates and controls the switch in polarity and how is this linked to adherens junction (AJ) disassembly? Epithelial cells frequently display polar plasticity through processes known as epithelial-to-mesenchymal transition (EMT) and its reverse, mesenchymal-to-epithelial transition (MET), which developing neurons appear to mirror. By highlighting recent work addressing these specific challenges in the developing cortex, spinal cord, retina and cerebellum, we will highlight the emerging idea that akin to epithelial cells, progenitors in the GZs of the CNS require the EMT-MET machinery to undergo a change in polarity that leads to their delamination, differentiation and maturation.

#### **DEVELOPING NEOCORTEX**

Development of the neocortex, with its unique lamination process, provides an attractive model to investigate the cellular remodeling essential for establishing the CNS (Rakic, 1971, 2009). Neural stem cells at the transient embryonic zone switch from a proliferative symmetric cell division phase to an asymmetric

phase, giving rise to radial glial cells (RGCs), the progenitor cells of the cortex (Bayer, 1986, 1990; Malatesta et al., 2000; Campbell and Götz, 2002; Nadarajah and Parnavelas, 2002; Götz, 2003). Successive waves of migration at the VZ form an insideout gradient of neurogenesis to establish the laminar cortical structure (**Figure 1B**; Angevine and Sidman, 1961). Stratification of the cortex requires radial and tangential migration of neurons, as shown by electron microscopy, lineage tracing and realtime imaging of brain slices (Mione et al., 1997; Wilson and Rubenstein, 2000; Marín and Rubenstein, 2001). Lamination of the cortex in CNS development requires precise spatial-temporal regulation of cortical migration (Métin et al., 2008). To guide the reader's appreciation and understanding of the morphogenetic changes that occur during corticogenesis, we will briefly discuss the cytoarchitecture of the newly formed RGCs.

Ultrastructure studies show that like epithelial cells, newly formed RGCs are morphologically polarized (Aaku-Saraste et al., 1996; Huttner and Brand, 1997; Chenn et al., 1998). They attach apically to the ventricular surface while extending long basal processes that span the entire cortical plate that affix at the overlying matrix produced by the pia. Apical anchoring is mediated by specialized intercellular adhesion complexes that involve cadherins, nectins, JAMs and β-catenin (Aaku-Saraste et al., 1996; Zhadanov et al., 1999; Manabe et al., 2002; Junghans et al., 2005; Kadowaki et al., 2007). These complexes link the cytoskeletal scaffolds and coordinate signaling pathways in neighboring cells while the pial attachment is established by integrins and possibly cadherins (Anton et al., 1999; Graus-Porta et al., 2001). Relevance of the cell-cell contact in establishing the radial glial scaffold at the apical surface and its role in

signaling networks such as reelin is highlighted by cortical defects observed in β1integrin and Dab1/Rap1/cadherin deficient mice (Graus-Porta et al., 2001; Franco et al., 2011). As in epithelial cells, Cdc42 and polarity complexes such as Par proteins and Crumbs complex (Crb, PALS, PATJ, Lin7) are essential in establishing and maintaining these AJs (**Figure 2**; Manabe et al., 2002; Cappello et al., 2006; Imai et al., 2006; Bultje et al., 2009). Nevertheless, as development proceeds the RGCs divide giving rise to a progeny that become committed to their fate and must migrate to occupy their respective positions in the cortical layers. It appears that the newly born neurons must first break their AJs to delaminate from the apical surface. Second, they must remodel their cytoskeletons to initiate movement out of the VZ. Remodeling of polarity is apparent from time-lapse imaging and electron microscopy that show migrating neurons adopting an intermediate multipolar state before reacquiring some of the features needed for glial– guided migration (Shoukimas and Hinds, 1978; Nadarajah et al., 2003). Remodeling of junctions and polarity in these newly born neurons closely resemble the sequence of events during EMT-MET in epithelial cells (**Figure 1B**; Nelson, 2009; Lamouille et al., 2014).

Given that apical anchoring and delamination are crucial to cortical arrangement there has been great focus on understanding the apical protein complexes that allow for this GZ exit; and have helped to define the roles of N-cadherin/Rap1, adaptor protein Afadin, Lis1, β-catenin, cdc42/GSK-3β and polarity protein Pard3/Notch in maintaining the apical cytoarchitecture (Bos et al., 2001; Ooshio et al., 2007; Bi et al., 2009; Severson et al., 2009; Yokota et al., 2010; Franco et al., 2011; Jossin, 2011; Jossin and Cooper, 2011). A recent report showed that cadherinbased adhesions also facilitate Notch signaling (Hatakeyama et al., 2014). While these studies have identified key components of the machinery involved in establishing the polarity and GZ occupancy, the overarching question of how these components might be regulated demands attention. Because fate commitment and delamination are sequential events, it seemed plausible that proneural genes might also have a role in regulating delamination. Actin reorganization by proneural factors such as Neurogenin 2 (Neurog2) and Ascl1, activating Rho GTPases Rnd2 and Rnd3, respectively, allows remodeling of the actin cytoskeleton by inhibiting Rho activity, thus linking neuronal commitment and migration (Ge et al., 2006; Heng et al., 2008; Pacary et al., 2011). Although they may provide the physical force needed for RGC exit from the GZ niche, they do not initiate delamination, as silencing of Rnd3 disrupts the distribution of β-catenin and N-cadherin at AJs, indicating that Rnd3 is essential for maintaining the integrity of the junctions (**Figure 2**; Pacary et al., 2011, 2013). Additionally, the physical forces can be effective only if the RGCs first attenuate their intercellular adhesion through dissolution of AJs, lose their apical-basal polarity and acquire a multipolar nature. In neural crest cells and epithelial cells, this phenomenon is regulated by EMT factors that promote loss of epithelial characteristics and acquisition of mesenchymal attributes (Thiery and Sleeman, 2006; Baum et al., 2008). Central to this process are the Snail, Slug and Zeb family of transcriptional repressors (Chaffer et al., 2007; Baum et al., 2008). From these systems and the fact that newly committed neurons express such regulators, one can extrapolate a possible molecular link between commitment and delamination.

In support of the above idea, Scratch 1 and Scratch 2, members of the Snail superfamily, are expressed upon neuronal fate commitment in neocortex, in which they trigger active disintegration of AJs by directly repressing expression of Ecadherin (**Figure 2**; Itoh et al., 2013). Additionally, classical genes in the EMT pathway, such as those encoding Occludin and Nephrins, also show downregulation accompanied by increased expression of Mmp19 (Thiery and Sleeman, 2006; Itoh et al., 2013). Notably, Scratch1 overexpression has no effect on neurogenesis, thus identifying it as a delamination-specific pathway. The existence of an EMT-like pathway in corticogenesis is further buttressed by the finding that another regulator of the pathway, Snail, is expressed in both radial precursors and newborn neurons during corticogenesis (Zander et al., 2014a,b). Snail's role in the neocortex is not limited to regulating AJs; it also helps promote the survival, proliferation and self-renewal of cortical progenitors.

#### **DEVELOPING SPINAL CORD**

The central question of what molecular mechanism controls AJ disassembly during migration has been addressed in developing motor neurons (MNs) of the spinal cord. Spinal cord development is a multistage process with distinct subtypes V0, V1, V2, V3, interneurons and MNs (**Figure 1C**; Alaynick et al., 2011). Convergent action of morphogens such as Shh and retinoic acid elicits a unique set of transcriptional networks and factors (e.g., Olig2) that specify different neuronal subtypes (Mukouyama et al., 2006; Briscoe and Novitch, 2008). Studies in Olig2-mutant mice showed that the forkhead proteins Foxp1, 2 and 4 are essential for specifying MN fate and for migration (Ferland et al., 2003; Dasen et al., 2008; Rousso et al., 2008; Palmesino et al., 2010). Rousso et al. investigated the role of Foxp proteins in chicken and mouse spinal cord, showing them to be components of a gene regulatory network that links and balances AJs with cell fate (Rousso et al., 2012). Increased expression of Foxp2 and Foxp4 inversely affected expression of N-cadherin and Sox2, leading to dissolution of AJs and ectopic neurogenesis in the VZ. Conversely, their loss maintained the precursors in the progenitor state (**Figure 2**). Such transcriptional regulation of N-cadherin and Sox2 might also regulate the size of the embryonic VZ niche by controlling delamination (Rousso et al., 2012). Decreased Sox2 and cadherin levels have also been demonstrated in neural crest cells before delamination (Zappone et al., 2000; Bylund et al., 2003; Graham et al., 2003; Bello et al., 2012). These findings, together with the documented role of forkhead proteins in maintaining AJs in other systems by regulating cadherins, reinforces the idea of an EMT-like signature in CNS development (**Figure 1C**; Mani et al., 2007; Rousso et al., 2012).

Besides transcriptional control of adhesion proteins, delamination of progenitors also involves a change in polarity and cellular architecture, recently addressed in a new light by Das and Storey. By using structured illumination microscopy in chick neural tube, they discovered a cellular mechanism called apical abscission that participates in actin-myosin–dependent remodeling of primary cilium (Das and Storey, 2014). Apical abscission at the VZ detaches progenitors and leads to loss of apical complex proteins, a process characterizing loss of apical polarity (Farkas and Huttner, 2008; Das and Storey, 2012). This study answered the question of how the VZ, unlike archetypal epithelium undergoing EMT, maintains its integrity. It seems plausible that apical abscission might provide the progenitors an efficient way to transiently change their polarity without substantially altering their transcriptional profiles. If basal progenitors remodel their primary cilia in preparation for delamination in the neocortex and for apical shedding of MNs that could explain how newly differentiated neurons lose and reacquire polarity during delamination and migration by remodeling their apical complex proteins.

#### **DEVELOPING RETINA**

Like other developing CNS structures, the retinal neuroepithelium is pseudostratified. During retinogenesis, polarized progenitors remain anchored to the apical-basal membrane by AJs and TJs while their fate is determined by interkinetic nuclear migration (**Figure 1D**; Frade, 2002; Baye and Link, 2007, 2008). Subsequent establishment of the retinal layers requires postmitotic detachment of retinal progenitors from the apical surface, reorganization of polarity and migration to the appropriate layers (Miyata, 2008).

In mouse retinal progenitors, polarity is imparted by two apical complexes, the partitioning defect (PARD) complex comprising Pard3, Pard6, aPKC and cdc42 and crumbshomologue (CRB) comprising Crb1-3, Pals1/MPP5 and Patj (Koike et al., 2005; Cappello et al., 2006; van de Pavert et al., 2007; Park et al., 2011; Alves et al., 2013; Dudok et al., 2013; Heynen et al., 2013). In mice lacking CRB2, early retinal progenitors show abnormal lamination due to greater proliferation of lastborn progenitors. Membrane-palmitoylated protein 3 (MPP3), a scaffolding cell-cell adhesion protein, is also localized to the retinal sub-apical region and interacts with Pals1 and Dlg1 (Pellikka et al., 2002; Alves et al., 2013). Perturbation of MPP3 in developing retina was found to cause focal disruption of AJs and ectopic cell localization (Dudok et al., 2013). The need to maintain polarity is further stressed by laminar defects in Pals1 conditional-knockout mice, in which mislocalization of neurons was found throughout the retina (Park et al., 2011). Additionally, dysregulation of aPKC and cdc42 lead to lamination defects (Koike et al., 2005; Heynen et al., 2013).

Structural and signaling components come together at neural junctions. In vertebrates, the polarized epithelium's apical junction proteins, such as cadherin, occludins, claudins and JAMs, interact on the cytoplasmic side of the cell with a variety of polarity and signaling molecules, including Pard3 and βcatenin, that connect them to the actin cytoskeleton (Tsukita and Furuse, 1999; Tsukita et al., 1999; Nishimura et al., 2004; Umeda et al., 2004; Perez-Moreno and Fuchs, 2006). In the retina, alteration of β-catenin causes loss of polarity and adhesion, cell detachment, ectopic migration and spatiotemporally perturbed retinogenesis (Brault et al., 2001; Fu et al., 2006). The role of apical complexes is exemplified in retinogenesis, in which they not only establish adhesion and polarity but act as important nodes for diverse signaling pathways (e.g., Notch, Wnt, Hippo, mTOR) essential for specifying retinogenesis. As supporting evidence, Ohata et al., showed that Crb complex and Notch form a positive feedback loop that maintains apicobasal polarity; disruption of feedback causes laminar defects (Ohata et al., 2011).

There are some interesting differences between retinal neuroepithelium and other developing CNS structures, one of which was highlighted by Ivanovitch et al. By combining mosaic labeling of single cells with 4D confocal imaging during optic evagination in zebrafish, they showed two discrete populations of cells: basally positioned cells and core cells involved in establishing the retinal neuroepithelium (Ivanovitch et al., 2013). Interestingly, live imaging showed that basally positioned cells undergo precocious polarization, while core cells remain mesenchymal until optic vesicle formation. The authors found that core cells that localize Pard3 in a polarized manner undergo a special MET using apical points for intercalation in CNS outpocketing. Furthermore, modulation of Pard6γb and Laminin leads to failure of optic vesicle evagination (Ivanovitch et al., 2013).

Clearly, the transition in polarity and morphology of retinal progenitors requires rearrangement of their cytoskeleton that generates physical forces required for movement. While this process is a quintessential requirement for determining the laminar structure of developing CNS, how are such processes regulated remains an open question. A chemical mutagenesis screen in medaka fish identified the guanine-nucleotide exchange factor ArhGEF18, a RhoA- and Rac-specific GEF factor, to be a key regulator of retinal architecture and function that also controls apicobasal polarity and proliferation (Herder et al., 2013; Loosli, 2013). In epithelial cells, small GTPases of the Rho family act as molecular switches that regulate and coordinate the actin cytoskeleton, cell junction assembly and polarity (Heasman and Ridley, 2008; Samarin and Nusrat, 2009). Additionally, Rho GTPases are regulated by transforming growth factor β (TGFβ), which degrades RhoA by phosphorylating Pard6 (Ozdamar et al., 2005). TGFβ also downregulates Pard3 expression while activating the EMT regulator Snail (**Figure 2**; Wang et al., 2008). As TGFβ is expressed during early retinal embryogenesis, it could plausibly play a role in regulating or guiding a similar process at that time.

#### **DEVELOPING CEREBELLUM**

The cerebellum is an attractive model for studies of CNS development, primarily because of its remarkably simple laminar organization, which consists of two principal neurons (cerebellar granule neurons (CGNs) and Purkinje cells) and minority interneuronal populations (Roussel and Hatten, 2011). Granule neuron progenitors (GNPs) arise along the rhombic lip, at the midbrain-hindbrain junction, and migrate dorsally over the cerebellar anlage (Wingate, 2001), while a stream of radially migrating progenitors arising from neuroepithelium form the other cerebellar cell types (Hallonet and Le Douarin, 1993; Gao

and Hatten, 1994). After GNPs migrate away from the rhombic lip, they proliferate under the guidance of external cues, such as Shh, to generate a second GZ called the external granular layer (EGL; Hatten, 1999). The remaining cerebellar histogenesis occurs mainly postnatally. This protracted development provides an excellent opportunity to study distinct intermediate stages of neurogenesis. After their proliferative phase, GNPs exit the cell cycle to form a layer of cells that migrate tangentially, then undergo a morphological transformation that initiates radial migration through the molecular layer (ML) along glial fibers to their final laminar position (Rakic, 1971; Edmondson and Hatten, 1987; Komuro et al., 2001). In the outer EGL, the GNPs have a round morphology with multiple short processes (Manzini et al., 2006). As they migrate to the inner EGL, their cytoskeleton is reorganized to form two short horizontal processes that elongate during their journey to the ML (**Figure 1E**). After a brief latency at the ML, they acquire an elongated spindle shape and a vertical process (Komuro et al., 2001).

The maturation of CGNs and their migration requires polarization, which was carefully examined in two studies delineating role of Par complex in this process. The first study showed that manipulation of Pard6α function, both *in vitro* and *ex vivo*, disrupts the actomyosin cytoskeleton and blocks radial migration of CGNs (Solecki et al., 2009). As expression of Pard3A polarity protein increases as the CGNs mature, the second study investigated its role in CGN migration (Famulski et al., 2010). Whereas loss of Pard3A impeded radial migration of CGNs, ectopic expression led to precocious migration. Further, Pard3A expression was shown to be regulated by E3 ubiquitin ligase seven in absentia homolog (Siah) 1 and 2 (**Figure 2**). Reciprocal expression of Siah and Pard3A in developing CGNs indicated that Siah negatively regulates Pard3A expression. This study also linked polarity proteins with adhesion of CGNs. In the epithelial cells, Pard3A binds to three members of the JAM family via its PDZ domain and recruits them to TJs to establish polarity (Ooshio et al., 2007). Utilizing live probe imaging, Famulski et al., demonstrated identical spatiotemporal expression of these proteins during CGN maturation and showed that JAM-C is necessary and sufficient for CGN exit from the EGL (Famulski et al., 2010). These results indicate that not only is cell adhesion crucial for guiding a neuron's migration path but the acquisition of adhesion by CGNs and loss of some of multipolar features reflects the MET-like process (**Figure 1E**). This is unique from other developing CNS structures where the newly born neurons lose polarity and require it upon reaching their final laminar position. A pertinent question that sequentially arises from these observations is how polarity is reorganized and how immature neurons initiate, implement and conclude this essential morphogenic event.

#### **SUMMARY**

Addressing the morphological changes observed in newly born neurons and its relevance in setting up the laminar structure of CNS raises some interesting questions. How the polar plasticity of newly born is regulated and what are the key factors that are involved in this process across the various developing CNS structures? Further in the developing cerebellum, how do the neurons born from the RGCs after adopting a multipolar migratory feature upon reaching the second GZ restore their polarity? Like the epithelial system is there a regulator whose expression temporally coincides with the CGNs reacquiring epithelial characteristics aka MET like processes. If so, what would be some of the targets of this MET like processes in developing neurons? Do these factors also play a role in differentiation and maturation of the neurons?

#### **ACKNOWLEDGMENTS**

We thank Sharon Naron for excellent editorial support in preparation of the manuscript and members of the Solecki lab for editing the manuscript. The Solecki Lab is funded by the American Lebanese Syrian Associated Charities (ALSAC), by grant #1-FY12-455 from the March of Dimes, and by grant 1R01NS066936 from the National Institute of Neurological Disorders and Stroke (NINDS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NINDS or the National Institutes of Health.

#### **REFERENCES**

deletion results in dramatic brain malformation and failure of craniofacial development. *Development* 128, 1253–1264.

Wang, X., Nie, J., Zhou, Q., Liu, W., Zhu, F., Chen, W., et al. (2008). Downregulation of Par-3 expression and disruption of Par complex integrity by TGF-beta during the process of epithelial to mesenchymal transition in rat proximal epithelial cells. *Biochim. Biophys. Acta* 1782, 51–59. doi: 10.1016/j.bbadis.2007.11.002

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 17 December 2014; accepted: 10 February 2015; published online: 04 March 2015*.

*Citation: Singh S and Solecki DJ (2015) Polarity transitions during neurogenesis and germinal zone exit in the developing central nervous system. Front. Cell. Neurosci. 9:62. doi: 10.3389/fncel.2015.00062*

*This article was submitted to the journal Frontiers in Cellular Neuroscience*.

*Copyright © 2015 Singh and Solecki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms*.

# Molecules and mechanisms that regulate multipolar migration in the intermediate zone

#### **Jonathan A. Cooper \***

Fred Hutchinson Cancer Research Center, Division of Basic Sciences, Seattle, Washington, USA

#### **Edited by:**

Takeshi Kawauchi, Keio University School of Medicine / Japan Science and Technology Agency, Japan

#### **Reviewed by:**

Eckart Förster, University of Hamburg, Germany Hidenori Tabata, Aichi Human Service Center, Japan

#### **\*Correspondence:**

Jonathan A. Cooper, Fred Hutchinson Cancer Research Center, Division of Basic Sciences, 1100 Fairview Ave N, Seattle Washington 98109, USA e-mail<EMAIL_ADDRESS>
Most neurons migrate with an elongated, "bipolar" morphology, extending a long leading process that explores the environment. However, when immature projection neurons enter the intermediate zone (IZ) of the neocortex they become "multipolar". Multipolar cells extend and retract cytoplasmic processes in different directions and move erratically sideways, up and down. Multipolar cells extend axons while they are in the lower half of the IZ. Remarkably, the cells then resume radial migration: they reorient their centrosome and Golgi apparatus towards the pia, transform back to bipolar morphology, and commence locomotion along radial glia (RG) fibers. This reorientation implies the existence of directional signals in the IZ that are ignored during the multipolar stage but sensed after axonogenesis. In vivo genetic manipulation has implicated a variety of candidate directional signals, cell surface receptors, and signaling pathways, that may be involved in polarizing multipolar cells and stabilizing a pia-directed leading process for radial migration. Other signals are implicated in starting multipolar migration and triggering axon outgrowth. Here we review the molecules and mechanisms that regulate multipolar migration, and also discuss how multipolar migration affects the orderly arrangement of neurons in layers and columns in the developing neocortex.

**Keywords: neuron migration, axonogenesis, multipolar migration, neocortex development, radial migration, cortical lamination, neuron locomotion, mini-columns**

#### **INTRODUCTION AND SCOPE**

The neocortex develops by the coordinated migration of projection neurons from the neocortical ventricular zone (VZ) and interneurons from the ganglionic eminences (Hatten, 2002; Marín and Rubenstein, 2003). While projection neurons move generally outwards from the VZ to the top of the cortical plate (CP), live imaging of individual cells has revealed that their radial progress is interrupted by an extended period of random migration. During this time the neurons appear to be "stellate" or "multipolar" (MP), characterized by multiple (>3) cytoplasmic projections that point in different directions. The primary cilium and centrosome are oriented randomly relative to the pial surface. MP cells migrate with frequent changes of direction, moving sideways (tangentially), up (towards the pia) or down (towards the VZ). Axons are initiated during the MP phase. MP neurons are quite different from migrating neurons in other brain regions, which generally are elongated in the direction of travel and have a prominent leading process. MP migration can lead to horizontal dispersion of neurons, and may be functionally significant for forming cortical circuits. In addition, the duration of MP migration differs from neuron to neuron, which has implications for cortical layering. Finally, the signaling mechanisms that initially cause polarized cells from the VZ to become MP, and which cause MP cells to resume radial migration and become bipolar, remain mysterious despite intense study.

This review addresses new developments and continuing uncertainty regarding the external signals and intracellular mechanisms that regulate MP cells at three important times: when MP migration starts, when the axon starts to grow, and when MP cells repolarize towards the pia and resume radial migration with bipolar morphology. The review also discusses the implications of MP migration for the radial unit hypothesis of cortical wiring and for cortical lamination. The focus is on newer research from the last 5–10 years, with an emphasis on loss of function studies. Readers are referred to excellent reviews for earlier work (Bielas et al., 2004; LoTurco and Bai, 2006; Ayala et al., 2007).

#### **BACKGROUND: PHASES OF MIGRATION OF NEOCORTICAL PROJECTION NEURONS**

Four phases of projection neuron migration have been described through detailed histological and live imaging studies (Shoukimas and Hinds, 1978; O'Rourke et al., 1992; Nadarajah et al., 2001; Hatanaka and Murakami, 2002; Tabata and Nakajima, 2003; Hatanaka et al., 2004, 2009; Noctor et al., 2004; Ochiai et al., 2007; de Anda et al., 2010; Namba et al., 2014; **Figure 1**). In phase 1, asymmetric division of radial glia progenitors (RG) in the VZ creates new post-mitotic neurons and intermediate progenitors (IP). These cells exit the VZ with bipolar or "pin-like" morphology. Phase 2 starts when cells reach the subventricular zone (SVZ)/IZ and become multipolar (**Figure 1**, stage 2A). MP IP divide in the SVZ and their daughters resume MP migration (stage 2A'). After a day or more in the MP phase, a ventricleor horizontally-oriented process near the centrosome begins to extend and becomes the axon (stage 2B). Phase 2 ends when the MP cell reorients the Golgi and centrosome towards the pia, establishes a dominant pia-directed leading process, and starts radial migration, trailing the axon behind (Hatanaka et al., 2004; de Anda et al., 2010) (stage 2C). This is known at the multipolar to bipolar (MP-BP) transition, and requires the stabilization of a dominant leading process and the correct orientation of that process towards the pia. After the MP-BP transition, neurons rapidly exit the IZ by locomotion along RG (phase 3), followed by phase 4, terminal translocation to the top of the CP.

The principal signaling pathways implicated in these major transitions are summarized in **Figure 1** and described in more detail below. Many additional molecules are required for the multipolar to bipolar transition but their regulation by external signals is unclear. These molecules are very briefly reviewed in **Box 1**.

## **THE START OF MP MIGRATION**

Neurons become multipolar at the boundary between the VZ and SVZ/IZ (Tabata and Nakajima, 2003; Noctor et al., 2004; **Figure 2**, stage 2A). It is not clear whether this transformation is active or passive. The BP-MP transition could be actively induced by signals present in the SVZ/IZ, but such signals have not been identified. Alternatively, physical interactions could induce the morphological change. The IZ contains a dense neuropil of horizontally-packed axons that may be a barrier to radial migration of BP cells. This region is also the stiffest part of the developing brain (Iwashita et al., 2014). Matrix stiffness influences the morphology and migration behavior of mesenchymal cells, and may similarly affect neurons (Roca-Cusachs et al., 2013).

#### **BOX 1 | GTPases, protein kinases and cytoskeletal proteins implicated in the transition from multipolar to bipolar migration.**

In addition to the principal signaling pathways discussed in the main text, the following genes and proteins are implicated in regulating the transition from MP migration to BP migration in the CP. It is not clear whether they are regulated by external signals: they may be cell-intrinsic, or controlled by a transcription program set in motion at the time of neurogenesis.

The protein kinase **Mst3/Stk25** regulates MP migration, apparently by inhibiting RhoA (Tang et al., 2014). Mst3-deficient cells accumulate in the IZ with rounded morphology. Mst3 contains a site for phosphorylation by Cdk5. Cdk5 phosphorylates Mst3 in vitro, and Mst3 phosphorylation is reduced in Cdk5-/- brain. Kinase-defective and non-phosphorylated mutants of Mst3 fail to rescue migration. This suggests that Cdk5 phosphorylation and activation of Mst3 is required for migration out of the IZ. In vitro, Mst3 over-expression reduces RhoA GTP loading. Remarkably, Mst3 directly phosphorylates RhoA and a non-phosphorylated RhoA mutant has increased GTP loading. This suggests that Mst3 directly inhibits RhoA. Overexpressed RhoA causes IZ arrest, and RhoA knockdown rescues Mst3-inhibited neurons, consistent with Mst3 activation by Cdk5 inhibiting RhoA and permitting exit from the IZ. However, it is not known whether Mst3 activity and RhoA phosphorylation change during migration.

**Rnd2** is an unusual GTPase that is regulated primarily by expression level. Rnd2 expression is induced soon after neurons leave the VZ by proneural gene Neurog2 and maintained in MP cells by NeuroD1 (Heng et al., 2008). Neurog2 and Rnd2 are required for the MP-BP transition, and the requirement for Neurog2 can be by-passed if Rnd2 is artificially expressed (Hand et al., 2005; Heng et al., 2008). The main function of Rnd2 appears to be as an inhibitor of **RhoA**. Accordingly, neurons lacking Neurog2 can be rescued by inhibiting RhoA. The results imply that Rnd2 inhibits RhoA during MP migration and high RhoA activity delays the MP-BP transition.

A requirement for inhibition of RhoA by Rnd2 seems to be in conflict with the requirements for activation by Mst3 and by PlxB2 (see main text) (Azzarelli et al., 2014). How can we reconcile these findings? It is possible that cycling of RhoA between GTP and GDP states is required, or activity be increased in some parts of the cell and inhibited in others. Alternatively, it is possible that some intermediate level of RhoA activity is permissive for radial migration.

**GSK3** is important for neuron polarization and axon growth and branching in vitro (Hur and Zhou, 2010). In bipolar neurons, activated GSK3β localizes the microtubule plus-end binding protein adenomatous polyposis coli (APC) to the distal ends of microtubules in the tip, allowing centrosomal forward movement and neuronal migration (Asada and Sanada, 2010). Morgan-Smith et al examined cortical lamination by use of mice lacking GSK3α and deleting GSK3β in early postmitotic neurons with NeuroD6- Cre (Morgan-Smith et al., 2014). While lower neuron layers were normal, upper layers were dispersed, suggesting defects in MP migration or locomotion along RG. When Cre was electroporated into the VZ at E15, GSK3-deficient neurons became arrested in the IZ with MP morphology. Axons were present, but branched abnormally. Phosphorylations of DCX at Ser327 and CRMP2 at Thr514 were inhibited. GSK3 also regulates the canonical Wnt signaling pathway, but mutation of β-Catenin or triple mutation of all Disheveled family members with NeuroD6-Cre did not lead to gross layering defects.

#### **BOX 1 | Continued**

Rab GTPases regulate membrane traffic. When endocytosis is inhibited with dominant-interfering **Rab5** or Rab5 knockdown, some neurons accumulate in the IZ with abnormal, rounded morphology (Kawauchi et al., 2010). However, other neurons had bipolar morphology but were stalled at the bottom of the CP with an abnormally thick trailing process. Co-culture experiments suggested that Rab5-inhibited neurons bind more tightly to RG. Rab5-inhibited cells have a small increase in surface NCad, and NCad knockdown partially rescues their migration. This suggests that abnormally high NCad after the MP-BP transition may inhibit RG-dependent locomotion.

In contrast with Rab5's role in endocytosis, **Rab11** regulates membrane recycling to the cell surface. Dominant-interfering Rab11 reduces NCad on the cell surface and increases NCad in recycling endosomes (Kawauchi et al., 2010). Neuron migration is also slowed, although the exact stage was not determined. If the delay is in the IZ, then the results would be consistent with Rab11 mediating the Reelin-dependent NCad exocytosis in the MP stage, and Rab5 mediating NCad endocytosis to allow locomotion.

**Cdk5** is a protein kinase that is related to cell cycle kinases but is expressed and functions in non-mitotic cells, including neurons. Cdk5 is required for the MP-BP transition (Ohshima et al., 2007). Cdk5-deficient cells extend axons but remain multipolar and retarded in the IZ. When cells do enter the CP, their leading processes are often branched, suggesting problems with stabilizing a single leading process (Hatanaka et al., 2004; Ohshima et al., 2007). A similar phenotype was noted for cells lacking the Cdk5 activator, p35 (Gupta et al., 2003). Cdk5 phosphorylates many proteins involved in MP migration, including DCX, Ndel1 and p27kip1 (reviewed by Ayala et al., 2007) and axin (Fang et al., 2011). A key question for Cdk5 is whether it is dynamically regulated by external signals during MP migration or is constitutively active. The serotonin 6 receptor, **5HT6R**, was recently reported to regulate MP exit (Jacobshagen et al., 2014). The authors suggested that 5HT6R may work through Cdk5, because 5HT6R binds to Cdk5 and the migration of 5HT6R-deficient cells was partly rescued by over-expressing Cdk5 and its activator, p35 (Jacobshagen et al., 2014). Unfortunately, there is no evidence that serotonin or other extracellular 5HT6R ligands regulate migration.

The role of the **Jnk1** pathway is controversial. Jnk1 activity, measured using antibodies to phospho-Jnk, is high in the IZ, where the Jnk1 activating kinase, **DLK** is highly expressed (Hirai et al., 2002; Kawauchi et al., 2003). Deletion of the DLK gene decreases Jnk1 activity, inhibits axonogenesis, and slows neurons at the IZ-MZ boundary (Hirai et al., 2006). DLK is a member of the Mixed Lineage Kinase family, and like other family members can be activated by Rac1. This suggests that Jnk may be activated by Rac1 via DLK. Indeed, dominant-interfering **Rac1** inhibits Jnk activation in vivo (Kawauchi et al., 2003). In turn, Rac1 may be activated by Rac1 GEFs **STEF** and **Tiam1** which are highly expressed in the IZ and CP (Kawauchi et al., 2003). Accordingly dominantinterfering STEF/Tiam1, dominant-interfering Jnk1, or Jnk inhibitors all stall neurons in the IZ, suggesting that a STEF/Tiam1-Rac1- DLK-Jnk1 pathway is involved in IZ exit. The phenotypes of the inhibited neurons are not consistent with a simple, linear pathway, however. Rac1-inhibited neurons are rounded, with reduced processes, suggesting a general defect in process extension and a failure of radial polarization. In contrast, Jnk1-inhibited neurons have undergone the MP-BP transition but the leading process is twisted and irregular (Kawauchi et al., 2003). This suggests that Rac1 regulates MP migration through several effectors, and that Jnk1 is involved in locomotion of BP neurons out of the IZ.

#### **BOX 1 | Continued**

A different conclusion was drawn by Westerlund et al., who found that **Jnk1** is an inhibitor, not an activator, of the MP-BP transition (Westerlund et al., 2011). They found that Jnk1 knockdown or gene deletion stimulates axon outgrowth, IZ exit and migration through the CP. The mechanism appears to involve the neuron-specific Stathmin family member **SCG10**. SCG10 stabilizes microtubules when it is phosphorylated by Jnk1. In vivo, SCG10 knockdown or expression of non-phosphorylated mutant SCG10 stimulates axon outgrowth and IZ exit (Westerlund et al., 2011). Tyrosinated (unstable) tubulin is increased in Jnk1-/- cortex. Expressing phosphomimetic mutant SCG10 in Jnk1 knockdown neurons restores the normal, slow, exit from the MP zone. This leads to a model in which Jnk1 phosphorylates SCG10, stabilizes microtubules and inhibits the microtubule remodeling required for axon outgrowth and for the MP to BP transition. It is not clear how to reconcile this study with those of Kawauchi et al. (2003) and Hirai et al. (2006).

**srGAP2**: srGAP2 contains an F-BAR membrane-bending domain and a GAP domain specific for Rac1. Knockdown of srGAP2 decreases the number of neurons in the IZ and lower CP, suggesting that endogenous srGAP inhibits IZ exit and BP locomotion through the CP (Guerrier et al., 2009). Rapidly migrating srGAPdeficient neurons in the CP have a less-branched leading process than normal. Endogenous srGAP2 may thus inhibit formation of a single leading process. Accordingly, over-expressed srGAP2, or just the F-BAR domain, inhibits the MP-BP transition. However, it is not known if srGAP2 activity changes during migration.

**Kinesin6**: Kinesin6 is a plus-end directed microtubule motor that also binds actin. Kinesin6 knockdown inhibits the MP-BP transition (Falnikar et al., 2013). Kinesin6 crosslinks and slides antiparallel microtubules in the mitotic spindle. Similarly, in differentiating neurons, kinesin6 helps establish and maintain antiparallel microtubules in the dendrites (Lin et al., 2012). In polarized migrating neurons, kinesin6 concentrates near the centrosome in the base of the leading process, potentially helping maintain mixed orientation microtubules in the leading process and concentrating actin in this region (Falnikar et al., 2013).

**FGF13** is an FGF-homologous family (FHF) member that is not secreted, but that acts inside the cell through mechanisms that are receptor-independent. Wu et al showed that FGF13 localizes in growth cones of cultured neurons, binds to microtubules, and stimulates microtubule polymerization (Wu et al., 2012). FGF13 deleted neurons have defective axon outgrowth in vitro. In vivo, FGF13 knockout delays MP neurons in the IZ, and those neurons that do enter the CP have an excessively branched leading process. Knockdown of FGF13 in utero causes a similar MP delay, which is rescued by wildtype FGF13 but not by a multi-alanine mutant that does not bind microtubules. In many ways, FGF13 resembles DCX, which is also a + end microtubule stabilizer that is enriched at the leading edge. Indeed, DCX knockdown causes IZ delay, which is partly rescued by FGF13 over-expression. Reciprocally, FGF13 knockdown is partly rescued by DCX over-expression, suggesting that DCX and FGF13 function in parallel.

Alternatively, cells may transform to MP morphology in order to weave between the axons that cross the radial path.

A third possibility is that proneural genes, expressed at the last RG division, induce the expression of proteins that cause pin-like cells to detach from the VZ and become multipolar (Itoh et al., 2013b). For example, induction of **Scratch** down-regulates **E Cadherin** (ECad, Cdh1), allowing separation of apical junctional

on processes that appear to be dynamically regulated by intrinsic transcription changes or signals from the environment. Reelin and neurotrophins BDNF and NT3 are shown as non-directional signals, while Sema3A is thought to form a gradient and provide a direction signal. TAG1 is a protein displayed on the surface of axons in the lower IZ. Abbreviations: junction dis., junction disassembly; actin reorg., actin reorganization; up arrow, increase; down arrow, decrease.

complexes (Itoh et al., 2013a). **p27kip1**, **Dcx** and **Rnd2** are other examples. Dcx and Rnd2 seem to be regulated by cell-intrinsic factors and are considered further in **Box 1**. p27 is considered further here because its expression may be dynamically regulated by signaling through gap junctions (Liu et al., 2012).

**p27** is well known for regulating the cell cycle, but it also inhibits the small GTPase **RhoA** and regulates microtubules, thereby increasing cell motility (Besson et al., 2004, 2008; Godin et al., 2012). p27 knockout inhibits neuron migration from the VZ to the IZ, which is rescued by a mutant that lacks the N-terminal, cell cycle kinase regulatory function but retains its C-terminal, RhoA-binding region (Nguyen et al., 2006). Surprisingly, p27 expression in MP cells requires **Connexin 43** (Cx43): p27 expression is inhibited by Cx43 knockdown and increased by Cx43 overexpression (Liu et al., 2012). Knockdown of Cx43 inhibits IZ entry, with a consequent decrease in MP cells (Elias et al., 2007; Liu et al., 2012). Cx43 is best known as a component of gap junctions, aqueous channels for cell-cell communication. In the CP, however, Cx43 provides adhesion between BP neurons and RG, thereby enabling locomotion (Elias et al., 2007). Empirically, one would not expect that MP entry would require cell-cell adhesion. Indeed, MP entry requires Cx43's channel function and C terminus, suggesting that gap junctions are involved (Liu et al., 2012). Cx43-dependent cell-cell communication could prime the p27 gene for induction during cell migration. The communication could occur between cells in the SVZ or between RG progenitors. Indeed, Cx43 is also needed for gap junction communication between progenitors and for interkinetic nuclear movement in the VZ (Liu et al., 2010). Therefore, gap junction communication between RG progenitors could be permissive for p27 gene induction by proneural genes when neuroblasts reach the IZ.

**p27** is also required later, for MP migration and exit from the IZ. A detailed study from Kawauchi et al. (2006) showed that acute knockdown of p27 with shRNA arrests neurons in the IZ. Cells in the lower IZ arrest with rounded morphology and reduced processes but those in the upper IZ have a clear radial process, suggesting defects in MP migration and locomotion of BP cells out of the IZ. The authors found that p27 inhibits phosphorylation of **cofilin**, an actin-severing protein that is inhibited by phosphorylation (i.e., p27 stimulates actin severing) (Kawauchi et al., 2006). This function of p27 is critically dependent on phosphorylation of p27 by **Cdk5** at Ser10, which protects p27 from proteasomal degradation and increases p27 levels. p27 likely inhibits cofilin phosphorylation by inhibiting **RhoA**, a known activator of the cofilin kinase, **LIMK** (Kawauchi et al., 2006). Therefore, Cdk5 and p27 together promote IZ exit by activating cofilin. Curiously, however, excess active cofilin also inhibits IZ exit (Kawauchi et al., 2006), suggesting that the balance between active and inactive cofilin is critical.

#### **AXON INDUCTION**

MP cells extend axons during the random migration phase, before they reorient towards the pia and begin radial migration (Hatanaka et al., 2004; Barnes and Polleux, 2009; Hatanaka and Yamauchi, 2013; Namba et al., 2014; **Figure 2**, stage 2B). Axon extension is first detected in the lower IZ, which contains corticofugal efferents (Hatanaka et al., 2009; Namba et al., 2014). This suggests that signals from corticofugal axons may induce or stabilize axons. Corticofugal axons express the homophilic adhesion molecule **TAG1** (Cntn2), which is absent from thalamocortical axons in the upper IZ (Fukuda et al., 1997). Nascent axons produced by MP cells also express TAG1 and align with the corticofugal axons (Namba et al., 2014). TAG1 knockdown in the MP neurons inhibits axon formation and inhibited radial migration. The functional domains of TAG1 that are required for homophilic binding in trans *in vitro* are also required cellautonomously for axon outgrowth. These results suggest that signals from TAG1 in the environment induce and stabilize the nascent axon by homophilic interactions. TAG1 is linked to the cell surface by a C-terminal glycosyl-phosphatidyl-inositol (GPI) moiety and is localized to lipid rafts, where it potentially activates Src-family kinases. Indeed, Namba et al show that the Src family kinase (SFK) **Lyn** and small GTPase **Rac1** are likely involved in stabilizing axons downstream of TAG1 (Namba et al., 2014).

In addition to TAG1, several receptor tyrosine kinases (RTKs) regulate axon induction *in vivo*. The neurotrophin receptors **TrkB** (Ntrk2) and **TrkC** (Ntrk3) and ligands **BDNF** and **NT3** are expressed in the VZ/SVZ. Trk inhibitors inhibit axonogenesis and migration out of the IZ in slice cultures (Ip et al., 2012). Also, sequestering neurotrophin ligands by expressing dominant interfering mutants of both TrkB and TrkC by *in utero* electroporation inhibited axon outgrowth and increased the percentage of MP neurons in the IZ (Nakamuta et al., 2011). TrkB knockout causes cortical lamination defects but axon defects were not noted, perhaps due to compensation by TrkC (Medina et al., 2004). Injection of BDNF or anti-BDNF antibodies into the ventricle stimulates or inhibits neuron migration, respectively (Fukumitsu et al., 2006). However, injected BDNF accelerates the entire neurogenic and migration process, so neurons born on a given day arrive more quickly at the top of the CP, settle in a lower layer position, and express earlier fate markers than usual. Taken together, these experiments suggest that neurotrophins probably stimulate axonogenesis and radial migration independently of neurogenesis, but cause and effect are not completely clear. Another RTK, **Kit**, is also expressed in migrating neurons and knockdown inhibits axon outgrowth and delays radial migration, although the mechanism remains to be determined (Guijarro et al., 2013).

Axon induction by BDNF *in vitro* requires **phospholipase C**γ, **calcium**, and calcium-calmodulin regulated kinase kinase (**CaMKK**α**; Nakamuta et al., 2011**). Dual inhibition of TrkB and TrkC or knockdown of phospholipase Cγ or CaMKKα inhibited axon outgrowth and radial migration *in vivo*, suggesting that signaling through phospholipase Cγ and CaMKKα is important. An alternative mechanism involves α2-chimerin (Ip et al., 2012). α2 chimerin is a Rho GAP (GTPase activating protein) that contains an SH2 domain through which it binds to BDNF-stimulated TrkB (Ip et al., 2012). α2-chimerin knockdown at E14 stalls neurons in the MP stage. Rescue experiments showed that migration requires the SH2 domain but not GAP activity. α2-chimerininhibited neurons have abnormally high levels of dephosphorylated (active) **CRMP2**, a protein that stabilizes microtubules. This suggests that α2-chimerin-stimulation of CRMP2 phosphorylation may regulate migration. Consistent with this, migration of α2-chimerin-deficient cells was rescued by over-expressing wildtype or phospho-mimetic CRMP2 but not a non-phosphorylated mutant. Other studies have shown that CRMP2 is needed for the MP-BP transition, consistent with the idea that CRMP2 phosphorylation is needed to remodel the microtubule cytoskeleton (Sun et al., 2010). However, axon formation requires **Cdk5** and **axin** to inhibit **GSK3**β and thereby reduce phosphorylation of CRMP2 (Fang et al., 2011). This implies that CRMP2 is dephosphorylated during axon formation and re-phosphorylated when a radial leading process is stabilized. Nevertheless, there is no evidence that CRMP2 phosphorylation state changes during axon formation or migration. Indeed, the immunoreactivity of phospho-CRMP2 relative to total CRMP2 is constant through the IZ and CP (Ip et al., 2012). More study of specific phosphorylation sites and subcellular localization of CRMP2 may help resolve the mechanism.

## **THE COORDINATION OF AXON INDUCTION WITH THE TRANSITION FROM RANDOM TO RADIAL MIGRATION**

The appearance of an axon before random migration ends suggests the possibility that axon formation is a prerequisite for starting radial migration (**Figure 2**, stage 2C). The TAG1 results are interesting in this regard. As mentioned above, TAG1 knockdown inhibits both axonogenesis and radial migration (Namba et al., 2014). Since TAG1 is an axonal molecule, it is unlikely to regulate radial migration directly. Therefore, this study suggests that axon outgrowth may be required before radial migration starts. In addition, inhibiting TrkB, TrkC and Kit impaired radial migration as well as axon induction (Fukumitsu et al., 2006; Nakamuta et al., 2011; Ip et al., 2012; Guijarro et al., 2013), consistent with, but not proving, causality.

On the other hand, there is evidence that neurons can enter the CP without axons. Deleting the kinase **LKB1** (Stk11, Par4) in progenitors inhibited axon outgrowth but neurons still migrated out of the IZ and into the CP, albeit in reduced numbers and with abnormal, highly branched leading processes (Barnes et al., 2007). LKB1 mutation does not cause gross lamination defects (Morgan-Smith et al., 2014). However, different results were obtained when LKB1 was acutely knocked down with shRNA (Asada et al., 2007; Matsuki et al., 2013). Many LKB1-deficient neurons accumulated in the IZ with multiple axon-like processes, suggesting defects in polarization which impact the selection of a single axon and migration (Asada et al., 2007). Acute knockdown of two LKB1 co-activators, **Stk25** and **STRAD**α, also inhibited axon outgrowth and delayed cells in the IZ (Matsuki et al., 2010, 2013; Orlova et al., 2010). However, germline knockout of Stk25 did not affect layering or axonogenesis (Matsuki et al., 2013). The results are consistent with important roles for Stk25-STRADα-LKB1 in axon outgrowth and radial migration, but there may be bypass mechanisms that come into play when these proteins are absent. Overall the question of whether axonogenesis is required before a multipolar cell can stabilize a pia-directed leading process and start radial migration remains unanswered.

## **Unc5D REGULATES THE TRANSITION TO RADIAL MIGRATION**

**Unc5D** is a co-receptor for **Netrins**, forming a complex with **DCC**/Unc40 and modulating DCC signaling. Unc5D expression is highest in the IZ (Sasaki et al., 2008). Transcription factor **FoxG1** represses Unc5D expression (Miyoshi and Fishell, 2012). FoxG1 is expressed in the VZ/SVZ and CP but is reduced in the IZ. Thus Unc5D increases when cells start MP migration and decreases when cells enter the CP. This suggests that the decline in Unc5D may trigger radial migration. However, inhibiting Unc5D expression (by over-expressing FoxG1) delayed the MP-BP transition, and co-over-expression of FoxG1 and Unc5D rescued normal migration. This suggests that the MP-BP transition requires high levels of Unc5D but the disappearance of Unc5D does not define the timing. In other systems, Netrin can bind to DCC in the absence of Unc5D, but the responses to DCC and Unc5D-DCC are different. DCC is expressed continuously during neuron migration. This means that MP cells have Unc5D-DCC complexes and Unc5D-deficient cells (which arrest in the IZ) have only DCC. This suggests that free DCC might inhibit IZ exit. Indeed, over-expressing DCC delays MP exit, which is rescued by co-expressing Unc5D (Miyoshi and Fishell, 2012). This suggests that MP exit may require DCC repression by Unc5D as a permissive event.

# **REELIN AND N-CADHERIN AS PERMISSIVE SIGNALS FOR RADIAL POLARIZATION**

**Reelin** is a secreted protein that regulates neuron migration in many brain regions (Honda et al., 2011). Full-length Reelin is most abundant where it is made in the MZ, but it is also cleaved and active fragments diffuse through the CP to the IZ (Jossin et al., 2007; Uchida et al., 2009). Genetic deletion of Reelin interferes with migration at several steps (Honda et al., 2011). Overexpressing dominant-negative Reelin receptors induces delay in the MP phase, with little or no inhibition of axon outgrowth (Jossin and Cooper, 2011).

Signaling proteins that are activated by Reelin are also required for radial polarization of MP cells. *In vitro*, Reelin stimulates a pathway including tyrosine phosphorylation of **Dab1** by **Src** and **Fyn**, recruitment of **Crk/CrkL** adaptors and **C3G**, a guanine nucleotide exchange factor (GEF) for **Rap1**, and increased GTP binding to Rap1 (Cooper et al., 2008). Some of these signaling proteins are also important in the MP-BP transition. For example, Dab1 knockdown inhibits migration in the IZ (Young-Pearse et al., 2007). Src and Fyn, the Crk/CrkL binding sites on Dab1, and the Crk and CrkL genes, are all required for normal lamination, but MP migration was not analyzed (Park and Curran, 2008; Feng and Cooper, 2009). C3G mutants arrest in the IZ with MP morphology and decreased Rap1GTP, implicating C3G and Rap1 (Voss et al., 2008). *In utero* electroporation with dominantinterfering Reelin receptors or Rap1A inhibitors also delays radial polarization, and over-expressed Rap1A partly rescues Reelininhibited MP cells, suggesting that Rap1A activation is necessary and partly sufficient for polarization (Jossin and Cooper, 2011). Rap1GTP regulates GEFs for the Ras-family member **RalA**, which regulates exocytosis, as well as Rho-family members **Rac1** and **Cdc42**, which regulate the actin cytoskeleton. Inhibition of Rac1or Cdc42 causes arrest of MP cells in the IZ (Kawauchi et al., 2003; Konno et al., 2005; Jossin and Cooper, 2011). Inhibition and rescue experiments implicated RalA, RalB, Rac1 and Cdc42 in Rap1-dependent radial polarization, suggesting that exocytosis and the actin cytoskeleton are involved (Jossin and Cooper, 2011).

The importance of exocytosis for radial migration is reinforced by the finding that a primary role of Reelin in the IZ is to induce surface traffic of **N-Cadherin** (NCad, Cdh2) (Jossin and Cooper, 2011). Rap1- or RalA-inhibited neurons have decreased cell-surface NCad and their migration is rescued by NCad over-expression. Dominant-interfering NCad inhibits MP exit (Kawauchi et al., 2010; Jossin and Cooper, 2011). The results suggest that Reelin regulates Rap1, RalA and other small GTPases to upregulate NCad, and that surface NCad is required to sense direction signals for radial migration and the MP-BP transition. NCad is not needed for locomotion of BP neurons, but is required again later, when Reelin regulates the final positioning of neurons at the top of the CP (Franco et al., 2011; Gil-Sanz et al., 2013).

It is unclear whether Reelin induces radial migration or is permissive. *In vivo*, NCad levels are high throughout the IZ, dependent on Reelin (Jossin and Cooper, 2011), so Reelin may induce NCad translocation to the cell surface throughout the period of MP migration. Moreover, Reelin is unlikely to be a direction signal: Reelin produced in the VZ or soaked into a brain slice can substitute for Reelin from the MZ (Magdaleno et al., 2002; Jossin et al., 2004). Therefore, Reelin may prime cells to sense another molecule that provides a direction signal and triggers radial migration as an instructive cue (Jossin and Cooper, 2011). Reelin is also required for correct radial orientation of other types of neurons (Landrieu and Goffinet, 1981; Nichols and Olson, 2010; Schneider et al., 2011; O'Dell et al., 2012), but in these situations it is also not clear whether it is instructive or permissive.

#### **SEMA3A MAY INDUCE AND ORIENT THE LEADING PROCESS**

**Plexin** (Plx) and **Neuropilin** (Nrp) family proteins are coreceptors for secreted **Semaphorins**, such as Sema3A. Importantly, Sema3A is secreted at the top of the CP and forms a gradient through the cortex. It is thus ideally suited to be a chemotactic factor for radial migration.

Seminal studies from Polleux and Ghosh showed that Sema3A orients cortical neurons so their dendrites grow towards the pia and axons towards the ventricle (Polleux et al., 1998; Polleux and Ghosh, 2002). Sema3A acts directly as an instructive signal by inhibiting axons and promoting dendrites (Shelly et al., 2011). Since the leading process of radially migrating neurons later develops into the dendritic tree, Sema3A may similarly induce the radial leading process on MP neurons. Indeed, Azzarelli et al found that MP neurons accumulate in the IZ when PlxB2 is inhibited (Azzarelli et al., 2014). Similarly, knockdown or Cremediated deletion of Nrp1, PlxA2, PlxA4 or PlxD1 increased the number of neurons trapped in the white matter and lower layers after birth, consistent with inhibition of MP migration (Chen et al., 2008). *In vitro*, Sema3A attracts neurons when added to one side of a slice culture or to one side of a porous membrane (Chen et al., 2008). This suggests that the gradient of Sema3A could provide a direction signal for radial migration, perhaps by stabilizing the leading process. However, it should be noted that cortical lamination is normal in Nrp1 mutant mice, at least up to E14.5 when embryonic lethality precludes analysis (Hatanaka et al., 2009). *In utero* expression of Nrp1/2 inhibitors at E12.5 or E15.5 also had no major effects on migration, although axon pathfinding was impaired (Hatanaka et al., 2009). It is not clear how to reconcile these findings.

The downstream signaling from Plx and Nrp receptors is currently unclear. However, PlxB2 may induce radial migration by stimulating **RhoA**. RhoA has a general role in stabilizing filamentous actin and inducing actomyosin contraction. PlxB2 binds to a RhoA activator, **PDZ-RhoGEF**/LARG. Removing PlxB2 lowered RhoA activity and over-expressing RhoA partly rescued the migration of PlxB2-deficient neurons. The C terminus of PlxB2 is required to bind PDZ-RhoGEF and rescue migration of PlxB2-deficient cells. This suggests that PlxB2 activates RhoA through PDZ-RhoGEF and that RhoA activity is needed for radial migration. However, there is currently no evidence that RhoA activity increases when radial migration starts. Cyclic nucleotides may also be involved (Liu et al., 2004). One attractive possibility is that Sema3A, coming from the top of the cortex, contacts the upper side of a multipolar cell, activates PlxB2, Nrp1 and RhoA locally, inducing and stabilizing a pia-directed process. However, testing this model will require improved RhoA activity reporters and high-resolution live-imaging of MP neurons as they reorient radially in the IZ.

## **OTHER SIGNALING MOLECULES REQUIRED FOR RADIAL MIGRATION: INSTRUCTIVE OR PERMISSIVE?**

The last decade has seen the discovery of a plethora of proteins that are required for or inhibit the MP-BP transition. In most cases, authors have used loss of function (knockdown or knockout) and gain of function (in most cases, over-expression) experiments to demonstrate necessity and sufficiency of specific proteins for radial migration. Some of the proteins, including LIS1 (Pafah1b1), Ndel1, dynamin, FilaminA and Doublecortin (DCX), were reviewed thoroughly previously, and will not be considered here (LoTurco and Bai, 2006; Ayala et al., 2007). Progress in understanding other genes that regulate radial migration is summarized in **Box 1**. However, in nearly every case, it remains unclear whether they are permissive or whether their activation (or inhibition) sets the start time for radial migration and transformation to BP morphology.

# **LATERAL MIGRATION OF MP CELLS AND THE RADIAL UNIT HYPOTHESIS**

Once MP migration starts, neurons migrate laterally as well as up and down. The horizontal movement needs to be reconciled with the radial unit hypothesis (Rakic, 2000). This postulates that cells move radially from the VZ to CP, allowing the protomap of functional areas in the VZ to be relayed into the CP. Radial migration means that sister neurons are arranged in "mini-column" units (reviewed by Gao et al., 2013). Consistent with strictly radial migration, locomoting neurons are generally detected within 1 or 2 cell diameters (10–20 µm) of their sister RG (Noctor et al., 2001, 2002). After migration is complete, neurons preferentially form electrical and chemical synapses with their sisters in other layers (Yu et al., 2009, 2012). If neurons wander laterally during MP migration, how do they remain close to their parental RG and how do they find their sisters when they are forming circuits?

One way that the MP cells could remain close to their parent RG would be to follow a random walk. Given a MP velocity of 2–4 µm/h (Tabata and Nakajima, 2003; Jossin and Cooper, 2011; Dimidschstein et al., 2013) and travel time of 24 h, a neuron could travel 50–100 µm in a straight line but just a few cell diameters (10–20 µm) if it changes direction every hour. However, the distance would be expected to increase with the time spent migrating. The relationship between horizontal spread and time spent migrating has not been established, but Sanada et al. (2004) found that the lateral dispersion of neurons was reduced from 16 µm to 4–8 µm when Reelin signaling was inhibited, even though the cells had presumably spent more time in the IZ and had progressed less far into the CP. This is the opposite of what would be expected if lateral dispersion is passive.

An alternative hypothesis is that the direction of MP migration is not random, but is controlled by signaling molecules. Indeed, there is now excellent evidence that horizontal movement of MP cells is regulated by Ephrin (Efn) and Eph family proteins (**Figure 3**).

Efns are cell surface proteins that activate Eph family RTKs on other cells and generate "forward signals". Reciprocally, an Eph can activate "reverse signaling" into an Efn-expressing cell. EfnA molecules are glycosylphosphatidylinositol-linked cell surface proteins that bind EphA family members. EfnB molecules are transmembrane proteins that bind EphB family members. Efns and Ephs regulate cell sorting in many developmental systems (Fagotto, 2014).

Several **EfnA** and **EphA** genes are expressed in the developing cortex (Torii et al., 2009). In the IZ, EfnAs are expressed more medially and less laterally. Their ligands, EphA RTKs, are expressed in the IZ in a counter gradient, higher laterally than medially. This expression pattern suggests that EfnA or EphA activity could regulate lateral dispersion, e.g., if cells of the same EfnA:EphA ratio sort together. This hypothesis was tested by studying a EfnA2/3/5 triple mutant (Torii et al., 2009). At birth, there were no gross effects on cortical development or layer order, but the cortical layers contained undulations. When clones were labeled at E12.5 and examined at E14.5, neurons in wildtype clones averaged 20 µm from the RG while they were only 10 µm distant in the triple knockout. Live imaging confirmed that tangential migration is inhibited when EfnAs are absent.

Somewhat paradoxically, over-expression of an EfnA ligand, EphA7 or EphA4, caused over-expressing neurons to segregate from non-expressing neurons in the IZ (Torii et al., 2009). Each cluster contained neurons from several clones. Later, these clusters gave rise to cortical columns of over-expressing cells separated from other columns by normal cells. Column formation required EphA cytoplasmic domains, suggesting forward signaling from EfnA to EphA. Live imaging showed that this increased clumping is actually due to greater dispersion of EphA over-expressing neurons while they are migrating in the MP zone. Thus, the live imaging is consistent with the EfnA triple knockout, in that decreased EfnA (and decreased EphA forward signaling) decreases

on the strengths of signaling from EphA-EfnA and EphB-EfnB signals. See

text for details and references.

lateral movement in the IZ and increased EphA expression (and presumably EphA forward signaling) increases lateral movement of MP cells in the IZ. The results are consistent with MP cells choosing their lateral position based on the strength of EphA forward signaling (**Figure 3**, compare blue and red routes in absence and presence of EphA signaling).

In contrast with EfnAs stimulating lateral movement by forward signaling, endogenous EfnBs may inhibit lateral movement by reverse signaling. Over-expression of **EfnB1** at E13 in post-mitotic neurons caused over-expressing neurons to segregate away from non-expressing neurons (Dimidschstein et al., 2013). Despite the marked change in lateral distribution, layering was normal. There was no evidence for increased homotypic adhesion. Rather, the MP cells had shorter processes, perhaps indicating reduced migration in the IZ. This effect required the extracellular and cytoplasmic domains of EfnB1, consistent with reverse signaling from EphBs. The C terminus of EfnB1 binds a Rho-family GEF, **P-Rex1**. The abnormal clustering induced by over-expressing EfnB1 required the EfnB1 C terminus, P-Rex1 and **Rac3**, a little studied Rho-family member that is a primary substrate for P-Rex1 and is implicated in opposing Rac1 and reducing neurite number (Dimidschstein et al., 2013). This suggests that high levels of EfnB1 activity reduce MP migration in the IZ, reducing lateral spread (**Figure 3**). Consistently, deletion of EfnB1 increased the number of MP cell neurites and the speed of movement in the IZ. However, lateral spread of deleted clones was only increased by ∼20%. It is possible that combined disruption of EfnB1 with EfnB2 and EfnB3 would give a more severe dispersion and confirm the importance of endogenous EfnB family members in regulating horizontal movement.

#### **THE VARIED DURATION OF MP MIGRATION MAY AFFECT LAMINATION**

Time-lapse recordings and birthdating studies indicate that MP migration lasts for at least a day in the mouse, but the timing is very heterogeneous (Noctor et al., 2004; Westerlund et al., 2011). Neurons that spend longer in the MP phase are expected to arrive later at the top of the CP than neurons that travel more rapidly. This means they will enter higher cortical layers. Since neuron fate is thought to be fixed at the last division of the RG progenitor, different transit times are expected to blur the discrete layering of neuronal subtypes that characterizes the mature cortex (Molyneaux et al., 2007; **Figure 4**). It remains unclear whether or how the varied timing of MP migration is compensated at other stages of development.

One major source of variability is that post-mitotic neurons and IP enter and leave the IZ at different rates (Tabata et al., 2009). Radial glia daughters (RGDs) that are post-mitotic move slowly (>10 h) from the VZ to the SVZ/IZ but have a relatively short MP phase, entering the CP between 36 and 60 h (**Figure 4**, see RGD1). In contrast, IPs move quickly (<10 h) to the SVZ/IZ but then divide. The intermediate progenitor daughters (IPDs, **Figure 3**) become multipolar and continue migrating. They enter the CP >60 h after the RG division (Tabata et al., 2009). IP daughters are thus expected to layer above their "aunts" (RG daughters) at the top of the CP (**Figure 3**). Does this mean that IP daughters switch

**FIGURE 4 | Different timing of intermediate zone exit influences cortical lamination** Neurons spend variable times migrating in the intermediate zone (IZ). Postmitotic neurons derived directly from radial glia (i.e., radial glia daughters, RGD) spend approximately 2 days in the IZ before entering the cortical plate (CP). Intermediate progenitors (IP) divide to create intermediate progenitor daughters (IPDs), for a total time of approximately 5 days in the IZ. As a result RGD born on day 0 (RGD1) arrive at the top of the CP 2–3 days before IPDs whose mother IP also left the VZ on day 0. As a result the IPDs layer above RGD1, and co-layer with RGD2, born 2 days later. If neuron fate is fixed at the last RG division, then this would cause mixing of neurons of different fates in the same layer.

to a later fate than RG daughters or does this lead to broadening and mixing of cortical layers?

One idea is that fates are not fixed at the last division of the RG progenitor but remain plastic (Fishell and Hanashima, 2008). Cell fate may be determined at the last division, whether it occurs in the VZ or SVZ/IZ. In this way, the fates of IP daughters and RG daughters entering the same lamina could be equivalent. Indeed, manipulation of FoxG1 not only delays cell exit from the IZ, so neurons shift to a higher lamina, but also adjusts the neuron fate to match the new position (Miyoshi and Fishell, 2012; Toma et al., 2014). Further testing of this hypothesis during normal development requires approaches for distinguishing IP daughters from RG daughters, and testing whether they co-layer and express the same markers (Tabata et al., 2009).

#### **CONCLUSIONS**

Amazing technical refinements in mouse genetics, imaging, *in utero* manipulation, and slice culture methods have led to the discovery of a large number of genes that regulate MP migration, axonogenesis, and direction of movement. Many of the genes have been subjected to epistasis analysis, implying causal relationships and sequences of contingent events. Some protein activities are regulated by external signals, while others may be "hard-wired" by cell intrinsic mechanisms or transcription changes. Despite dramatic progress, it is still unclear how these different processes fit together. We have limited understanding of how different pathways interact and exactly when and where in the cell signaling occurs. Analysis is extremely challenging given the asynchrony of the cell population, the small size of the cells, and the difficulties of imaging signaling events within living tissue. However, given the continuous development of new technology, we can be optimistic that these obstacles will be overcome.

#### **ACKNOWLEDGMENTS**

I am grateful to Drs. Goichi Miyoshi, Yves Jossin, Gord Fishell and Nancy Ip for discussion during preparation of this review. The selection of papers and interpretation is my own, however, and I apologize to authors for papers omitted due to lack of space. Research in the author's laboratory is supported by research grants R01-NS080194 and R21-NS089888 from the U.S. Public Health Service.

### **REFERENCES**

cortex development. *J. Neurosci.* 31, 13613–13624. doi: 10.1523/jneurosci.3120- 11.2011

**Conflict of Interest Statement**: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 06 October 2014; accepted: 29 October 2014; published online: 14 November 2014*.

*Citation: Cooper JA (2014) Molecules and mechanisms that regulate multipolar migration in the intermediate zone. Front. Cell. Neurosci. 8:386. doi: 10.3389/fncel.2014.00386*

*This article was submitted to the journal Frontiers in Cellular Neuroscience*.

*Copyright © 2014 Cooper. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms*.

# Cellullar insights into cerebral cortical development: focusing on the locomotion mode of neuronal migration

#### Takeshi Kawauchi 1, 2, 3 \*

<sup>1</sup> Department of Physiology, Keio University School of Medicine, Tokyo, Japan, <sup>2</sup> Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, Saitama, Japan, <sup>3</sup> Laboratory of Molecular Life Science, Institute of Biomedical Research and Innovation, Foundation for Biomedical Research and Innovation, Kobe, Japan

The mammalian brain consists of numerous compartments that are closely connected with each other via neural networks, comprising the basis of higher order brain functions. The highly specialized structure originates from simple pseudostratified neuroepithelium-derived neural progenitors located near the ventricle. A long journey by neurons from the ventricular side is essential for the formation of a sophisticated brain structure, including a mammalian-specific six-layered cerebral cortex. Neuronal migration consists of several contiguous steps, but the locomotion mode comprises a large part of the migration. The locomoting neurons exhibit unique features; a radial glial fiber-dependent migration requiring the endocytic recycling of N-cadherin and a neuron-specific migration mode with dilation/swelling formation that requires the actin and microtubule organization possibly regulated by cyclin-dependent kinase 5 (Cdk5), Dcx, p27kip1, Rac1, and POSH. Here I will introduce the roles of various cellular events, such as cytoskeletal organization, cell adhesion, and membrane trafficking, in the regulation of the neuronal migration, with particular focus on the locomotion mode.

#### Edited by:

Thomas Knöpfel, Imperial College London, UK

#### Reviewed by:

Zoltan Molnar, University of Oxford, UK Gunnar P. H. Dietz, Schwabe Pharma Deutschland, Germany

#### \*Correspondence:

Takeshi Kawauchi, Laboratory of Molecular Life Science, Institute of Biomedical Research and Innovation, Foundation for Biomedical Research and Innovation, 2-2 Minatojima-Minamimachi Chuo-ku, Kobe 650-0047, Japan<EMAIL_ADDRESS>
> Received: 08 May 2015 Accepted: 22 September 2015 Published: 07 October 2015

#### Citation:

Kawauchi T (2015) Cellullar insights into cerebral cortical development: focusing on the locomotion mode of neuronal migration. Front. Cell. Neurosci. 9:394. doi: 10.3389/fncel.2015.00394 Keywords: microtubule, actin cytoskeleton, endocytosis, JNK, Rab5, Rab11, Rab7, Rap1

# Introduction

The brain is divided into many compartments, such as nuclei, layered structures, and cortical areas, allowing highly organized role allocations. The systematically allocated neuronal populations are generated from spatially restricted regions, the ventricular, and subventricular zones. Therefore, a long-distance migration from the ventricular side to the final destination is essential for constructing a functional brain. In line with this, defects in neuronal migration are associated with various neurological disorders (Gleeson and Walsh, 2000; Kawauchi and Hoshino, 2008). Several types of cortical malformations, including lissencephaly, double cortex syndrome (subcortical band heterotopia) and periventricular heterotopia (PVH), are thought to result from neuronal migration defects. These cortical malformations are frequently associated with intellectual disability and intractable epilepsy (Francis et al., 2006; Moon and Wynshaw-Boris, 2013; Reiner and Sapir, 2013; Lian and Sheen, 2015).  Furthermore, suppression of genes related to dyslexia (e.g., DCDC2, KIAA0319), autism spectrum disorder (ASD) (e.g., Auts2, CNTNAP2) and schizophrenia (e.g., SDCCAG8) disturbs neuronal migration, although it is unclear whether the neuronal migration defect is the main cause of the pathogenesis of these neurological and psychiatric disorders (Hannula-Jouppi et al., 2005; Kamiya et al., 2005; Meng et al., 2005; Paracchini et al., 2006; Wang et al., 2006; Kähler et al., 2008; Peñagarikano et al., 2011; O'roak et al., 2012; Zhang et al., 2013a; Hori et al., 2014; Insolera et al., 2014; La Fata et al., 2014) (**Figure 1B**). Interestingly, in addition to these genes, several environmental factors, such as stress and inflammation, are also associated with cortical development, including neuronal migration (Stolp et al., 2012; Hashimoto-Torii et al., 2014; Ishii and Hashimoto-Torii, 2015).

In the developing cerebral cortex, neuronal migration consists of several contiguous steps (Nadarajah and Parnavelas, 2002; Cooper, 2014; Takano et al., 2015) (**Figure 1A**). Newly generated neurons exhibit multipolar morphology in the lower part of the intermediate zone. The multipolar neurons form an axon and a leading process almost coincidentally with retraction of other processes. The bipolar-shaped neurons are called "locomoting neurons," which migrate along radial glial fibers with unique morphological changes (the locomotion mode of neuronal migration) (**Figure 2A**). At the final phase of neuronal migration, they switch over from the "locomotion" into a "terminal translocation."

Most studies so far have focused on the mechanisms of the morphological changes at the early phase of neuronal migration, including the multipolar-to-bipolar transition (Kawauchi and Hoshino, 2008; Heng et al., 2010). One reason is that many cortical malformation-related gene products are involved in the multipolar-to-bipolar transition. Second, the acquirement of neuronal polarity, a key step of neuronal maturation, occurs nearly simultaneously with the multipolar-bipolar transition. Third, suppression of cytoskeletal proteins or kinases often leads to defects in the early phase of migration due to the occurrence of various morphological changes at this stage.

In contrast to the early phase of migration, our knowledge of the locomotion mode is relatively poor. However, recent morphological and cell biological analyses have uncovered unique features of locomoting neurons. In this review, I will introduce recent advances in the molecular and cellular biology of neuronal migration with particular focus on the locomotion mode.

# The Early Phase of Neuronal Migration

Several steps of the early phase of neuronal migration, including the multipolar-to-bipolar transition, are required for the formation of the morphologies of the locomoting neurons. The formation of a leading process requires c-jun N-terminal kinase (JNK) (Kawauchi et al., 2003). Filamin A (FLNA), an actin-binding protein, is also involved in the early phase of migration, possibly including the leading process formation (Nagano et al., 2004). Knockdown of Lis1, a regulator of the dynein complex, a microtubule minus end-directed motor, suppresses the multipolar-bipolar transition (Tsai et al., 2005). FLNA and Lis1 have been identified as causative genes for PVH and lissencephaly, respectively, and both knockout of FLNA and Lis1 heterodeficiency show neuronal migration defects (Hirotsune et al., 1998; Zhang et al., 2013b).

In addition, many other molecules are reported to regulate the formation of multipolar morphology (e.g., Cdk5, p27kip1 , Arx, Rab5) (Kawauchi et al., 2006, 2010; Friocourt et al., 2008; Friocourt and Parnavelas, 2010) and multipolar-to-bipolar transition (e.g., Cdk5, PHF6, FMRP) (Ohshima et al., 2007; Zhang et al., 2013a; La Fata et al., 2014; Franzoni et al., 2015) at the early phase of migration (**Figure 1A**).

# The Locomotion Mode of Neuronal Migration