DOI 10.1515/hsz-2013-0277      Biol. Chem. 2014; 395(5): 465–476

Review Bhavin Shah and Andreas W. Püschel*

In vivo functions of small GTPases in neocortical development Abstract: The complex mammalian cortex develops from a simple neuroepithelium through the proliferation of neuronal progenitors, their asymmetric division and cell migration. Newly generated neurons transiently assume a multipolar morphology before they polarize to form a trailing axon and a leading process that is required for their radial migration. The polarization and migration events during cortical development are under the control of multiple signaling cascades that coordinate the different cellular processes involved in neuronal differentiation. GTPases perform essential functions at different stages of neuronal development as central components of these pathways. They have been widely studied using cell lines and primary neuronal cultures but their physiological function in vivo still remains to be explored in many cases. Here we review the function of GTPases that have been studied genetically by the analysis of the embryonic nervous system in knockout mice. The phenotype of these mutants has highlighted the importance of GTPases for different steps of development by orchestrating cytoskeletal rearrangements and neuronal polarization. Keywords: cortical development; knockout mouse; neuronal polarity; neuronal progenitor. *Corresponding author: Andreas W. Püschel, Institut für Molekulare Zellbiologie, Westfälische Wilhelms-Universität, Schloßplatz 5, D-48149 Münster, Germany, e-mail: [email protected] Bhavin Shah: Institut für Molekulare Zellbiologie, Westfälische Wilhelms-Universität, Schloßplatz 5, D-48149 Münster, Germany

Introduction In many cellular processes, GTPases play a central role as molecular switches that cycle between an active GTPbound and inactive GDP-bound state. As regulators of cell polarity, migration, cytoskeletal dynamics and intracellular trafficking they also perform essential functions in neuronal differentiation (Fukata et al., 2003; Govek et al., 2005; Heasman and Ridley, 2008; Iden and Collard, 2008;

Hall and Lalli, 2010). Neurons are highly polarized cells with a single long axon and multiple dendrites. This polarized architecture is essential for their function to process and transmit information. A key route to understanding the differentiation of neurons, therefore, is the search for signaling pathways that direct the establishment of their characteristic polarity. Primary cultures of dissociated cortical or hippocampal neurons have enabled the identification of many factors (including several GTPases) that are required for the formation of axons and dendrites (Barnes and Polleux, 2009). The differentiation of neurons in culture can be subdivided into five stages (Figure 1). After dissociated neurons attach to the culture substrate, they first develop lamellipodia (stage 1) before several immature neurites emerge that have a highly active growth cone at their tip (stage 2). These short neurites all have the potential to become an axon and undergo repeated periods of extension and retraction. At the transition from stage 2 to stage 3, one of these neurites starts to extend rapidly to become the axon (stage 3) while the other neurites begin to mature into dendrites at stage 4. These dendrites eventually form dendritic spines and synapses (stage 5). However, only a very small number of factors that affect axon formation in primary cultures have been tested for their in vivo function.

Cortical development The mammalian nervous system with its six cortical layers develops from a single sheet of cells that are highly polarized along their apico-basal axis and proliferate rapidly (Breunig et al., 2011). This pseudostratified neuroepithelium is transformed into a tissue with multiple layers by cell proliferation, asymmetric cell division and cell migration. The neuroepithelial cells serve as neuronal precursor cells, which generate successive wave of postmitotic neurons that form the cortical plate (CP) (Del Río et  al., 2000). The neuroepithelium matures into radial glial cells (RGCs) that line the ventricular zone and are the major source of pyramidal neurons in the dorsal telencephalon

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466      B. Shah and A.W. Püschel: GTPases in cortical development

Figure 1 Stages of neuronal polarization in culture. A few hours after attachment to the substrate, dissociated hippocampal neurons first form lamellipodia and filopodia (stage 1) before they extend multiple short neurites of similar length (stage 2). Upon polarization, one of these minor neurites is selected as the axon and extends rapidly (stage 3). The remaining minor neurites differentiate into dendrites (stage 4) that mature to form dendritic spines and synaptic contacts (stage 5) (based on Govek et al., 2005).

(Hartfuss et al., 2001; Anthony et al., 2004). RGCs undergo symmetrical divisions for expansion and self-renewal and asymmetrical divisions to generate daughter cells, which are either postmitotic neurons or basal progenitors (also called intermediate progenitors; Figure 2) (Anthony et al., 2004; Noctor et  al., 2004). Basal progenitors reside in the subventricular region, where they divide to give rise to neurons (Kowalczyk et al., 2009). Newly born neurons leave the VZ to form the different cortical layers. Successive mitoses give rise to waves of neurons that bypass earlier born neurons and occupy more superficial cortical layers (Gupta et  al., 2002; Gao et  al., 2013). Projection neurons from the CP extend their axonal processes below the CP in the intermediate zone (IZ) that contains relatively few somata except for the migrating neurons. The projection neurons assemble into the characteristic six layers by migrating radially in an inside-out pattern, wherein the earliest born neurons form the deepest layers and the latest born neurons occupy the superficial layers. The major characteristic that distinguishes the apically located RGCs in the VZ (also called apical progenitors) from the basal progenitors in the SVZ is an apical domain marked by the par-complex proteins Par-3, Par-6, aPKCλ/ζ, Cdc42 and the N-cadherin dependent adherens junctions (AJs) between the RGCs (Cappello et  al., 2006; Imai, 2006; Costa et  al., 2008; Bultje et  al., 2009; Marthiens and ffrench-Constant, 2009; Zovein et  al., 2010). Their long processes span the whole cortex and their glial endfeet attach to the basement membrane of the pial surface via integrin-mediated adhesion (Schmid and Anton, 2003). The most superficial layer beneath the basement membrane (layer 1) is occupied by the Cajal-Retzius cells (CR), which arise from the cortical hem and migrate

into the cortex during development (Meyer, 2010). They secrete the extracellular matrix protein reelin that plays a pivotal role in regulating neuronal migration and cortical organization (Ogawa et  al., 1995). Reeler mice that lack reelin show an inversion in the order of the cortical layers (Tissir and Goffinet, 2003). However, genetic ablation of the cortical hem that leads to an almost complete absence of CR cells does not interfere with cortical lamination, indicating that other sources of reelin can substitute for the missing CR cells (Yoshida et al., 2006). The signaling pathway activated by binding of reelin to its receptors VLDL receptor (VLDLR) and APOE receptor 2 (APOER2) leads to the phosphorylation of the cytoplasmic adaptor protein disabled 1 (Dab1) and activation of Src family kinases (Bock and Herz, 2003). Phosphorylated Dab1 recruits the adaptor proteins Crk and CrkL that promote phosphorylation of the Rap1 GEF C3G and the activation of Rap1 GTPases (Tissir and Goffinet, 2003; Herz and Chen, 2006; Frotscher, 2010). Genetic inactivation of VLDLR/ APOER2, Dab1, the kinases Src and Fyn, or Crk/CrkL results in similar defects in neuronal migration and cortical lamination with inverted cortical layers typical for the reeler cortex (Trommsdorff et al., 1999; Ballif et al., 2003; Kuo et al., 2005; Park and Curran, 2008). The development of the CP with its six layers requires the polarization of postmitotic neurons to generate a leading process for their migration along the RGCs and a trailing axon (Barnes et  al., 2008). Advances in imaging techniques have allowed observation of the behavior of neurons during their directed migration. Neurons born in the VZ initially move into the SVZ where they assume a multipolar morphology by extending multiple processes of similar length that dynamically extend and retract

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B. Shah and A.W. Püschel: GTPases in cortical development      467

Figure 2 Function of GTPases during cortical development. During early stages of cortical development, RGCs (red) serve as the main progenitors for the generation of projection neurons (Barnes and Polleux, 2009). They are connected by AJs at the apical surface and their basal processes attach to the basement membrane via integrin-mediated adhesions. The asymmetric division of RGCs gives rise to neurons (yellow) or basal progenitors (also called intermediate progenitors, blue). Basal progenitors divide symmetrically in the SVZ to generate neurons (not shown). Postmitotic neurons transiently assume a multipolar morphology (yellow) in the SVZ. Eventually, they become bipolar (green) by extending a leading process and a trailing axon to migrate radially along the glial processes (red). Once they reach their destination in the CP, they switch to a glialindependent somal translocation. At an early stage of development, Arl13b is required in neuronal progenitors to maintain their apicobasal polarity (1). Cdc42 and RhoA control apico-basal polarity and AJs of RGCs (2). Rac1 mainly regulates the proliferation and survival of neuronal progenitors (3). Rap1 is required in multipolar cells for their radial migration and for axon formation (4). Loss of Cdc42 also interferes with axon formation. Rnd2 functions during the transition from the multipolar to the bipolar phase of migration. Rnd3 regulates the bipolar migration of neurons by inhibiting RhoA signaling (5). Rab GTPases are important for the directed migration of neurons by regulating N-Cadherin trafficking. The glial-independent somal translocation is under the control of the reelin signaling cascade (reelin is secreted by Cajal-Retzius cells shown in pink) that includes Rap1 (6). CP, cortical plate; IZ, intermediate zone; MZ, marginal zone; SVZ, subventricular zone; VZ, ventricular zone.

(Tabata and Nakajima, 2003; LoTurco and Bai, 2006; Hatanaka and Yamauchi, 2013). Unpolarized stage 2 neurons in culture may correspond to this multipolar stage (Lewis et  al., 2013), suggesting that at least some of the factors identified in neuronal cultures may regulate the

polarity of migrating neurons in vivo. One of the processes eventually becomes the axon when the neurons reach the middle of the IZ. Subsequently, the cells become bipolar with a leading and a trailing process. Once the neurons are polarized, they initiate their radial migration using the support of glial processes (Nadarajah et al., 2001; Nadarajah and Parnavelas, 2002). Initially, it was assumed that the trailing process oriented towards the ventricular zone becomes the axon. More recent live cell imaging studies demonstrated that frequently a process which is oriented tangentially in the IZ is selected as the axon (Hatanaka and Yamauchi, 2013; Sakakibara et  al., 2013). When approaching the marginal zone (MZ), migrating neurons attach their leading processes to the MZ and switch from the glial-dependent to a glial-independent mode of migration called terminal or somal translocation (Nadarajah et al., 2001). Eventually, the leading process matures into a dendrite while the trailing axon continues to extend in the IZ. This complex behavior with distinct modes of migration involves different molecular machineries that work together to regulate the transition between the successive stages of neuronal differentiation during cortical development. Small GTPases, their guanine nucleotide exchange factors (GEFs) that activate them and GTPase activating proteins (GAPs) that inactivate them are key regulators of these developmental programs (Heasman and Ridley, 2008).

The role of GTPases The Ras superfamily of small GTPases can be subdivided into five subfamilies: the Ras, Rab, Ran, Arf/Arl, and Rho GTPases (Wennerberg et  al., 2005). The Ran GTPases regulate nuclear import and export of proteins as well as mitotic spindle assembly and nuclear envelope formation and will not be discussed here (Wennerberg et  al., 2005). A detailed review of all GTPases and their regulators that have been implicated in neuronal development based on studies in cultured cells is beyond the scope of this review. Excellent reviews are available that summarize these results (Heasman and Ridley, 2008; Iden and Collard, 2008). A large number of studies using cultured rodent hippocampal neurons identified important roles for small GTPases and their upstream regulators in neuronal differentiation and polarity (Iden and Collard, 2008; Hall and Lalli, 2010) but few have been analyzed genetically in mammals for their physiological function. In Table 1, we summarize the GTPases that have been shown to play a role in the development of the neocortex either

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– Loss of apico-basal polarity of RGCs. – No defects in glial polarity and scaffolding.

 

Foxg1-Cre Nestin-Cre, Gfap Cre

Phenotype

Arl13b

  – Disruption of AJs in RGCs.   – Conversion of RGCs to basal progenitors.   – Disruption of AJs in RGCs.   – Loss of apico-basal polarity of RGCs.   – Increased apoptosis.   – Disruption of neuronal polarity.   – Increased apoptosis.   – Axon guidance defects: commissural axons fail to cross the midline.   – Reduced survival and proliferation of neuronal progenitors.   – Delay in the radial migration of neurons   – Axon guidance defects: commissural axons fail to cross the midline.   – Delay in the radial migration of neurons.   – Impaired neuronal migration and axon formation in CGNs.   – No developmental defects in the cortex.   – Subtle defects in the development of the hippocampus.   – Cell non-autonomous defect in neuronal migration.   – Disruption of AJs in RGCs.   – Decrease in axon growth.   – Delay in neuronal polarization.   – Aqueductal stenosis leading to hydrocephalus.   – Defects in early steps of neurogenesis, defects in maintenance of RGC AJs.  – Defects in neuronal migration.   – Defects in migration due to N-Cadherin mislocalization.   – Postnatal defects in myelination.   – No developmental defects.   – Migration defects.  

Emx1-Cre

Inactivation

    Foxg1-Cre       Nestin-Cre     Foxg1-Cre       Emx1-Cre     Nestin-Cre   Knockout   Syn1-Cre, Knockout   Emx1-Cre     Gene trap     Knockout   Knockdown     Knockdown, dominant-negative constructs  Nestin-Cre   Knockout   Rap1 GAPs, dominant-negative constructs  

 

              Rac1     .         Rac3   Rac1; Rac3   RhoA     Rnd3/RhoE           Rab5, Rab7, Rab 11   Rheb1   Rheb2   Rap1  

Cdc42

GTPase

Table 1 GTPases that have been implicated in cortical development regulating at different stages of development.

Kawauchi et al., 2010 Zou et al., 2011 Zou et al., 2011 Franco et al., 2011; Jossin and Cooper, 2011 Higginbotham et al., 2013 Higginbotham et al., 2013

Xi Lin et al., 2013 Pacary et al., 2013; Pacary et al., 2011

Peris et al., 2012

Tahirovic et al., 2010 Corbetta et al., 2005 Corbetta et al., 2009 Cappello et al., 2012

Kassai et al., 2008

Chen et al., 2007; Leone et al., 2010

Garvalov et al., 2007

Chen et al., 2006

Cappello et al., 2006

References

468      B. Shah and A.W. Püschel: GTPases in cortical development

B. Shah and A.W. Püschel: GTPases in cortical development      469

by the use of RNA interference or by brain-specific conditional knockouts. In our review, we will focus on those GTPases that have been validated genetically for their role in the developing mouse brain. The value of genetic approaches to elucidate the physiological function of GTPases is highlighted by the analysis of the Rab GTPases Rab3A–D (Schluter et  al., 2004). These were thought to play an essential function in synaptic membrane traffic and neurotransmitter release. The analysis of quadruple knockout mice lacking all four Rab3 genes revealed that these GTPases modulate the release machinery but are not required for synaptic vesicle exocytosis per se. GTPases regulate cell morphology by coordinating different signaling cascades and feedback loops in response to extracellular and intracellular cues. Small GTPases have been extensively characterized biochemically and in cultured cell lines. GTPase function is often analyzed using constitutively active and dominant-negative mutants that lock them in the GTP- (e.g., RasV12) or GDP-bound states (e.g., RasN17), respectively (Yoshimura et al., 2006b). The dominant-negative GTPase mutants act by sequestering their GEFs, which blocks the activation of endogenous GTPases. These mutants allow for circumvention of the redundancy of highly homologous GTPases like RhoA–C encoded in the genome. However, the use of these mutants is not without problems because many GEFs are not selective for a single GTPase. An analysis of a conditional Cdc42 knockout indicates that dominant-negative mutants do not necessarily inhibit only the function of the corresponding endogenous GTPase (Czuchra et al., 2005). Dominant-negative Cdc42 (Cdc42N17) was reported to inhibit cell migration in an in vitro wound closure assay. However, Cdc42 knockout fibroblasts did not show defects in this assay while expression of Cdc42N17 in these knockout cells resulted in a reduction of wound closure speed, which demonstrates that this phenotype depends on the inhibition of GTPases other than Cdc42. The genetic analysis of GTPase function often requires the generation of nervous system-specific knockouts because of their essential function in embryonic development. Several transgenic lines are used to obtain these conditional knockouts by driving the expression of Cre recombinase to mediate the deletion of the target gene flanked by loxP sites at different time points of neuronal development. Most commonly used are Emx1-, Foxg1-, Nestin- and Nex-Cre. Emx1-Cre expression starts at around 9.5 days of gestation (embryonic day 9.5: E9.5), Foxg1-Cre at E9 and Nestin-Cre around E10.5 (Guo et al., 2000; Hebert and McConnell, 2000; Dubois et  al., 2006). Although the Nestin-Cre-mediated recombination commences as early as E10.5, complete recombination is achieved only

perinatally in contrast to Emx1-Cre where about 90% of recombination is achieved by as early as E12.5 (Liang et  al., 2012). A significant decrease in the volume of neocortex, hippocampus and striatum was reported for Foxg1-Cre heterozygous mice that were maintained on the C57BL/6J background, which makes this line less useful unless properly controlled for their genetic background (Eagleson et al., 2007). All these Cre lines mediate recombination in neuronal precursors and subsequently in the neurons arising from them. Nex-Cre-mediated recombination is restricted to pyramidal neurons, dentate gyrus mossy and granule cells and a subset of neuronal progenitors and is used to study the neuron-specific functions of genes (Wu et al., 2005; Goebbels et al., 2006).

Rho GTPases Rho GTPases have been implicated in the regulation of neurogenesis, neuronal differentiation and migration regulating cytoskeletal dynamics, cell shape and migration (Yoshimura et al., 2006a; Heasman and Ridley, 2008; Hall and Lalli, 2010). They are the most extensively studied group with respect to their function in the remodeling of the cytoskeleton and the establishment of polarity during cortical development. Most of the studies have focused on Rho (RhoA–C), Rac (Rac1–3), Cdc42 and to some extent Rnd1–3 (Rnd3 is also called RhoE) while much less is known about the other members of this subfamily (RhoQ/ TC10, RhoJ/Tcl, RhoD, RhoF/Rif, RhoU/Chp/Wrch1, RhoV/ Wrch2, RhoG, and RhoH). Rho, Rac, Cdc42 and Rnd1–3 are highly expressed during cortical development in the VZ and SVZ, already suggesting an important role in progenitors (Pinto et al., 2008). Cdc42 was identified as a central regulator of neuronal polarity first by knockdown experiments in cultured hippocampal neurons (Schwamborn and Püschel, 2004). It has been inactivated at different time points of development by Cre-mediated deletions, revealing different aspects of its function (Cappello et al., 2006; Chen et al., 2006; Garvalov et al., 2007; Peng et al., 2013). Deletion of Cdc42 by Emx1-Cre-mediated recombination leads to the gradual loss of the Par polarity complex and AJs from the apical membrane of RGCs while the attachment of the basal processes to the pial basement membrane is unaffected (Cappello et al., 2006). As a consequence, the short apical processes retract from the ventricular surface and RGCs divide in a more basal position. Molecular makers showed a slow conversion of RGCs into basal progenitors. Thus, the main function of Cdc42 during neurogenesis is

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470      B. Shah and A.W. Püschel: GTPases in cortical development the regulation of the Par polarity complex and AJs, which maintain the fate of RGCs as apical progenitor cells. Deletion of Cdc42 using Foxg1-Cre leads a similar result and abolishes the apical localization of Par6, aPKC, E-cadherin, β-Catenin and Numb in the neuroepithelium at E9.5 to E11.5 (Chen et al., 2006). In addition, the extension of radial glia fibers is severely compromised in this mutant, demonstrating that Cdc42 is essential to maintain the apico-basal polarity of RGCs. A Nestin-Cre-mediated deletion of Cdc42 results in defects in axon formation indicative of a loss of neuronal polarity in cortical neurons (Garvalov et al., 2007). Neurons cultured from Cdc42 knockout cortices develop minor neurites but the formation of axons is severely compromised. Anterograde tracing of axons with DiI revealed a severe reduction of axon formation in the embryonic cortex at E16.5. Only short axonal bundles remain present in this mutant, which may indicate an incomplete inactivation of the gene by Nestin-Cre. This conditional knockout also showed a considerable reduction in the thickness of the cortex because of increased apoptosis (Peng et al., 2013). Axon formation has not been analyzed in the Cdc42 knockout mediated by Emx1-Cre but it can be expected that it also has severe defects in neuronal polarity in addition to the other phenotypes described above. So far, Cdc42 is the only GTPase shown to be required for neuronal polarity in vivo. Together, the phenotypes of these mutants confirm that Cdc42 plays an important role in establishing and maintaining polarity in RGCs and neurons. The role of Rac1 in cortical development was first analyzed using the Foxg1-Cre line (Chen et al., 2007). Loss of Rac1 in neuronal progenitors does not affect axon formation but leads to errors in axon guidance. Rac1 mutants show defects in the formation of commissures and a failure of commissural axons to cross the midline. Similar results were obtained with an Emx1-Cre-mediated deletion of Rac1 (Kassai et al., 2008). In both knockouts, only mild defects in cortical lamination were observed, which did not disrupt the organization of cortical layers. Further analysis of the Rac1 knockouts revealed a function in maintaining the proliferation and survival of neuronal progenitors (Leone et  al., 2010). The size of the cortex is reduced in the Foxg1-Cre Rac1 knockout because of increased apoptosis of progenitors in the VZ and SVZ. Loss of Rac1 reduces the proliferation of the basal progenitors in the SVZ leading to an increased exit from the cell cycle and premature differentiation. By E14.5, Rac1 mutants no longer show a separate SVZ and progenitors fail to pause and proliferate in the SVZ. Tbr2-positive progenitors also show a precocious differentiation into neurons. However, the role of Rac1 in maintaining proliferation is restricted

to the progenitors in the SVZ and does not affect RGCs, indicating that other molecules are involved in regulating their fate and survival. This could involve Cdc42 as summarized above. In addition, a detachment of RGC endfeet from the basement membrane and an invasion of neurons through the basement membrane (BM) were observed in the Rac1 mutant. Unlike in the cortex, Rac1 is the only Rac GTPase expressed in the cerebellum, which allowed the identification of additional functions for Rac1 using the Nestin-Cre line (Bolis et al., 2003; Tahirovic et al., 2010). In cerebellar granule neurons (CGNs), Rac1 is required for migration, axon formation and survival (Tahirovic et al., 2010). Rac1deficient CGNs show impaired neuronal migration and axon formation both in culture and in vivo. Loss of Rac1 impairs the actin cytoskeleton in the growth cone and disrupts the formation of lamellipodia. This defect could be linked to the Rac1 effector WASP that enables actin remodeling during axonal growth. Like Rac1, Rac3 is also highly expressed in the developing cortex but a Rac3 knockout does not show any phenotype indicating a possible redundancy with Rac1 (Corbetta et al., 2005). Conditional deletion of Rac1 in the Rac3 knockout mice using a transgenic line that drives Cre expression by the synapsin I promotor (SynI-Cre) revealed subtle defects in the hippocampus (Corbetta et  al., 2009). Since SynI-Cre is first detectable only at E12.5, additional functions of Rac1 and Rac3 in neuronal development and polarization may be revealed in a Rac1:Rac3 double knockout that inactivates Rac1 at an earlier time point. Experiments in neuronal cultures have indicated an important role for Rho GTPases in neurite extension (Govek et al., 2005). Of the three GTPases RhoA, B, C, only RhoA has been analyzed genetically in the nervous system (Cappello et  al., 2012). Conditional deletion of Rhoa in the cerebral cortex by Emx1-Cre results in the formation of a distinct ‘double cortex’ phenotype that resembles neuronal migration defects associated with mutations in genes encoding microtubule-associated proteins like Dcx or Lis1 (Cappello et al., 2012). In humans these mutations are associated with the formation of a double cortex where a band of neurons is located in the white matter appearing as a subcortical band heterotopia (Bielas et al., 2004). In addition, neurons migrate beyond layer 1 and breach the BM, a phenotype resembling cobblestone lissencephaly. Although it has been shown that RhoA signaling is required for neuronal migration (Nguyen et  al., 2006; Pacary et al., 2011), the defects observed in the conditional RhoA knockout using Emx1-Cre are non-cell autonomous. RhoA-deficient neurons do not show any defects when they migrate in a wild type cortex indicating that RhoA is

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B. Shah and A.W. Püschel: GTPases in cortical development      471

dispensable in neurons but has an essential role in maintaining the RGC scaffold (Katayama et al., 2011; Cappello et al., 2012). Loss of RhoA in RGCs causes profound defects in the apical AJs and a destabilization of actin fibers and microtubules. The disorganization of RGCs is accompanied by an increased proliferation and reduced cell cycle exit of neuronal progenitors (Katayama et al., 2011). The atypical Rho GTPases Rnd1–3 form a distinct subgroup that shows little intrinsic GTPase activity (Foster et al., 1996). Rnd proteins, like other Rho GTPases, have also been implicated in regulating actin dynamics. Rnd3 is important for neurite extension in PC12 cells (Talens-Visconti et al., 2010) while Rnd1 is involved in axon extension in cultured neurons by regulating microtubule stability (Li et  al., 2009). During cortical development, Rnd2 is involved in the transition from the multipolar to the bipolar stage in the IZ while Rnd3 is required later during the migration of bipolar neurons in the CP (Pacary et al., 2011). Knockdown of Rnd2 and Rnd3 by in utero electroporation indicated that both GTPases promote neuronal migration by inhibiting RhoA signaling at distinct steps of migration. Knockdown of Rnd3 also disrupts the apical attachment of RGCs, interferes with interkinetic nuclear migration and changes the cleavage plane of their division (Pacary et al., 2013). The loss of Rnd3 also increases the proliferation of basal progenitors. Rnd3 performs its various functions by regulating actin filaments and most aspects of the Rnd3 knockdown phenotype can be rescued by expressing a constitutively active cofilin mutant. A Rnd3 gene trap mutant that lacks detectable Rnd3 protein shows postnatal lethality probably caused by neuromuscular defects (Mocholi et al., 2011). Histological analysis of this mutant did not reveal major differences between wild type and mutant cortex but hippocampal neurons showed a delayed polarization and reduced axon growth in culture (Peris et al., 2012). A targeted knockout of Rnd3 results in aqueduct stenosis and development of congenital hydrocephalus caused by an increased Notch signaling and an enhanced proliferation of ependymal cells (Xi Lin et  al., 2013), a phenotype that was not described for the gene trap mutant. The difference in the phenotypes described for the Rnd3 mutants is surprising and requires further investigation but may be accounted for by differences in the genetic background. A further analysis of the targeted Rnd3 knockout at earlier stages than investigated so far (Xi Lin et  al., 2013) may also reveal additional defects. Rnd3 was originally shown to competitively inhibit RhoA function in COS-7 cells by binding to ROCK-I (Riento et al., 2003). In cortical neurons, however, Rnd3 antagonizes RhoA not through effects on ROCK-I but by interacting with the Rho GAP p190RhoGAP (Pacary et al., 2011). The

mechanism of RhoA inhibition by Rnd2 remains to be elucidated in vivo (Pacary et al., 2011). The inhibition of RhoA by Rnd GTPases is one example for the different mechanisms that mediate a crosstalk between GTPases during neuronal migration. The function of Rnd1 and 2 in cortical development remains to be analyzed genetically.

Ras GTPases The Ras subfamily of GTPases regulates a variety of signaling processes linked to transcription, cellular differentiation and proliferation. It contains in addition to the well-known H-Ras, K-Ras and N-Ras, also R-Ras, TC21, M-Ras, RalA/B, the Rap GTPases (1A, 1B, 2A, and 2B), Rheb1/2, Rin and Rit. The three highly homologous Ras proteins H-Ras, N-Ras, and K-Ras appear to perform redundant function as a complete knockout of Hras or Nras and the combined knockout of both genes do not show a phenotype (Esteban et  al., 2001). By contrast, knockout of Kras results in early embryonic lethality, indicating that it performs essential functions and is sufficient for a normal development (Esteban et  al., 2001). R-Ras and H-Ras are also involved in axon specification in cultured neurons acting upstream of PI3K and GSK3β (Yoshimura et al., 2006b; Oinuma et al., 2007). However, the lack of studies addressing their function specifically in cortical development using knockout mice leaves their physiological role unclear. The GTPases Rheb1 and Rheb2 are activators of the highly conserved serine/threonine kinase mTOR, which is a central regulator of cell growth and metabolism (Wullschleger et al., 2006; Laplante and Sabatini, 2012). Different co-factors associate with mTOR to form two mTOR complexes (TORC) with distinct functions. TORC1 stimulates cap-dependent translation while TORC2 regulates the actin cytoskeleton by phosphorylating several kinases including Akt and Protein Kinase C isoforms (Facchinetti et al., 2008; Thoreen et al., 2012). Germline deletion of Rheb1 results in embryonic lethality whereas deletion of Rheb2 has no obvious phenotype (Zou et al., 2011). After conditional inactivation of Rheb1 using Nestin-Cre, no obvious developmental defects in brain structure were found except for a postnatal reduction in myelination. Inactivation of the Rheb1 target mTORC1 by conditionally deleting the gene for the mTORC1-specific subunit Raptor (gene symbol: Rptor) using Nestin-Cre leads to a decrease in neuronal cell size and number but does not affect the overall organization of the brain (Cloetta et al., 2013). The inactivation of Rptor revealed

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472      B. Shah and A.W. Püschel: GTPases in cortical development that mTORC1 signaling is important for gliogenesis, which begins during late cortical development at around E17.5, but seems to have minimal effects on neurogenesis. Conditional deletion of the gene for the mTORC2-specific component Rictor (gene symbol: Rictor) also leads to a reduction in neuronal cell size and a disruption of their morphology (Thomanetz et al., 2013). Especially the dendritic arbors of cerebellar Purkinje cells were severely disrupted. The disparity of phenotypes for Rheb1 and Rheb2 on the one hand, and Rptor and Rictor on the other hand, probably indicates that Rheb1 and Rheb2 perform redundant functions in the nervous system. Only a conditional knockout of both Rheb1 and Rheb2 using Emx1-Cre will reveal the full scope of functions during neuronal development. The Rap1 GTPases have been studied in neurons for their role in migration and polarity and were shown to play multiple roles in neuronal development (Franco et  al., 2011; Jossin and Cooper, 2011). They are encoded by two highly homologous genes: Rap1a and Rap1b. Rap1B is required for polarity in cultured hippocampal neurons downstream of PI3K and upstream of Cdc42 and Par3 (Schwamborn and Püschel, 2004). Using knockdown, dominant-negative constructs and overexpression of Rap1GAPs, Rap1 GTPases were implicated in regulating neuronal migration at multiple stages (Franco et al., 2011; Jossin and Cooper, 2011). During the polarization of multipolar neurons in the cortex, active Rap1 is required to orient neurons towards the CP and initiate their radial migration. Rap1 acts by increasing the level of N-cadherin at the cell surface through unknown mechanisms. However, the migration of bipolar neurons appears to be independent of Rap1 (Jossin and Cooper, 2011). Once neurons have reached the CP, Rap1 is involved in the somal translocation of neurons downstream of the reelin signaling pathway (Franco et  al., 2011). The defect in terminal migration after inactivation of Rap1 could be rescued by expressing N-cadherin indicating that it is the main downstream target of Rap1 at multiple stages of migration in the developing cortex. Further genetic analyses are still required to dissect the exact roles of Rap1 and other Ras GTPases during cortical development. Our analysis of Rap1a;Rap1b conditional knockout mice using Emx1-Cre revealed an essential function of Rap1 GTPases for RGC organization and neuronal polarity (B.S. and A.W.P., unpublished results). Neurons from Rap1a;Rap1b;Emx1-Cre embryos that are deficient for Rap1 GTPases show severe defects in neuronal polarity both in culture and in vivo resulting in the loss of axons in the cortex and the hippocampus. These results reveal an essential role of Rap1 GTPases that act redundantly

to regulate the polarization of RGCs and neurons during embryonic development.

Rab, Arf, and Arl GTPases The Rab and Arf/Arl GTPases regulate endo- and exocytosis and intracellular trafficking. The Rab family is the largest group of GTPases with more than 60 members in mammals (Pfeffer, 2001). Mutation of Rab GDP-dissociation inhibitor 1 (GDI1) in humans results in mental retardation, indicating that Rab GTPases play important functions in neurons (D’Adamo et  al., 1998). One function during neuronal migration involves the regulation of vesicle trafficking and endocytosis (Kawauchi et al., 2010). Suppression of different Rab GTPases in the cortex using dominant-negative constructs and in utero brain electroporation revealed that Rab5 and Rab11 are involved in neuronal migration in the cortex by regulating the trafficking of N-cadherin and its level at the cell surface. In addition, a Rab7-dependent lysosomal degradation pathway is also required for terminal migration and apical dendrite maturation. However, with few exceptions Rab GTPases have not been analyzed genetically in knockout mice. The mammalian Arf/Arl family of GTPases consists of 29 members: 6 Arfs, Sar1 and over 20 Arf-like proteins (Arls) that have been grouped together according to sequence homology and structural similarity (Kahn et al., 2006). Arf GTPases are involved in regulating membrane trafficking, but the exact in vivo role of Arls still remains largely unexplored. Arl3, Arl6, and Arl13b have been associated with vertebrate ciliopathies (Zhang et  al., 2013). Arl3-/- and Arl13b-/- mice are embryonic lethal. So far, only Arl13b has been shown to play a role in cortical development using brain-specific conditional knockouts (Higginbotham et al., 2013). Arl13b is localized to primary cilia and plays a major role in organizing the glial scaffold during cortex formation. Foxg1-Cre-mediated conditional deletion of Arl13b leads to an almost complete inversion of RGC apico-basal polarity and cortical organization. The somata of RGCs are localized close to the pial surface and not adjacent to the ventricle in the Arl13b mutant. Consequently, neuronal progenitors divide at the pial surface instead of the ventricular surface causing the inward migration of the neurons. The analysis of conditional knockouts induced at different time points using several Cre lines suggests that Arl13b is required early during the formation of RGCs from the neuroepithelium but not later during RGC expansion by symmetrical divisions. However, the molecular mechanism of

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B. Shah and A.W. Püschel: GTPases in cortical development      473

how the loss of Arl13b disrupts the RGC scaffold remains unknown.

Perspectives The genetic analysis of only a limited number of GTPases has already highlighted their important functions in neuronal development. As most of the work on GTPases in the developing cortex has been done using knockdown or dominant-negative constructs, many aspects of their function remain to be analyzed genetically to determine their role in vivo. As the mammalian genome often contains several closely related GTPases, the generation of double or even triple knockouts may be required to fully

elucidate their in vivo function. GTPases act in complex networks of signaling pathways and show crosstalk with other GTPases to control the function of polarity proteins like the Par polarity complex or cadherins and integrins. In the future, it will also be important to study the crosstalk of GTPases, how they are regulated in vivo and how they coordinate downstream signaling events to orchestrate the complex cellular processes shaping the nervous system. Acknowledgments: The analysis of Rap1 function was supported by the DFG (SFB 629). Received November 19, 2013; accepted December 24, 2013; previously published online January 4, 2014

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Bhavin Shah studied Biology at the Universities of Mumbai and Pune (India). He is currently doing his PhD at the University of Münster. His PhD project deals with the role of Rap1 GTPases and their upstream regulators in neuronal polarity and cortical development.

Andreas Püschel studied Biology at the Universities of Bonn and Heidelberg. During his PhD he worked from 1986 to 1989 with Peter Gruss first at the ZMBH in Heidelberg and then at the Max Planck Institute for Biophysical Chemistry in Göttingen on the regulation of Hox genes. After postdoctoral studies on Pax genes in zebrafish at the Institute of Neuroscience in Eugene (Oregon), he joined the Max Planck Institute for Brain Research in Frankfurt in 1992, where he investigated the role of semaphorins as axon guidance molecules. In 2001, he was appointed Professor in Molecular Biology at the Institute for Molecular Cell Biology at the University of Münster. His current research focuses on identifying and analyzing the signals that direct the differentiation of neurons using knockout mice and live cell imaging.

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