Cell-Autonomous Roles of ARX in Cell Proliferation and Neuronal Migration during Corticogenesis

ARX inactivation using RNA interference

A previous study that used Arx knock-out mice documented a number of brain defects (Kitamura et al., 2002). However, because it has been difficult to differentiate between primary and secondary effects of ARX inactivation, we decided to apply the RNAi technique to investigate the role(s) of this gene in corticogenesis. A major advantage of this method is that it allows us to explore cell-autonomous defects in a wild-type background. Thus, we designed three RNAi sequences (called S1–S3) that target three different regions within ARX mRNA (Fig. 1A). To test the effectiveness of our shRNA constructs on ARX expression, we transfected cells dissociated from the GE of E16 rat embryos. As a control, we used a mutated shRNA directed against the 3′UTR of DCX (3UTR3mhp) (Bai et al., 2003; Friocourt et al., 2007). Cells were fixed 48 h after transfection and immunolabeled with anti-ARX antibody. We observed that all cells transfected with the control shRNA expressed ARX in the nucleus as previously described (Poirier et al., 2004), but the vast majority of cells expressing the S1, S2, or S3 shRNAs showed reduced ARX immunolabeling (Fig. 1B). Similar results were obtained when COS-7 cells were transfected with both a mouse Arx construct, expressed under a CMV promoter, and different shRNAs. Cells were lysed 48 h after transfection and immunoblotted using anti-ARX antibody (Fig. 1C). Most of the ARX protein was absent compared with control cells that were transfected with triple point mutant shRNAs (S1m–S3m) (Fig. 1A), or the shRNA targeting the 3′UTR part of DCX, respectively (Fig. 1C,D).

To quantify the efficiency of our shRNAs on the stability of ARX mRNA, we used the psiCheck-2 vector (Promega). This vector contains both a synthetic version of Renilla luciferase reporter gene to monitor RNAi activity and a synthetic firefly luciferase gene that permits normalization of changes in Renilla luciferase expression to the expression of firefly luciferase. We cloned arx mouse cDNA into this vector, downstream to the synthetic Renilla luciferase gene and its translational stop codon. When this construct is transfected into a mammalian cell line, a fusion of the Renilla gene and ARX is transcribed. Functional Renilla luciferase is then translated from the intact transcript. If a specific shRNA binds to ARX mRNA and effectively initiates the RNAi process, the fused Renilla-ARX mRNA sequence is degraded, resulting in reduced Renilla luciferase activity. COS-7 cells were transfected with psiCheck2-ARX and the different shRNAs at a ratio 1:4. Forty-eight hours after transfection, Renilla and firefly luciferase activities were measured. Figure 1E shows the Renilla luciferase measurements normalized to the firefly luciferase ones. ARX expression was knocked down from ∼53% for S3 to 74% for S2. Together, these results show that all three shRNAs efficiently target ARX mRNA and thus reduce its expression. However, because S3 shRNA was the least efficient of the three, we did not use it in further experiments.

ARX affects progenitor cell proliferation

To examine the effects of ARX silencing on cell proliferation, S2 shRNA or a control shRNA (S2m or DCX 3UTR3mhp) were introduced together with a DsRed vector into neural progenitor cells of mouse neocortex by in utero electroporation at E13.5. We found that ARX inactivation resulted in a marked decrease, sometimes even a complete absence, of DsRed-positive cells in the VZ 3 d after transfection (Fig. 2A,B). To establish that this effect was not caused by increased cell death, we labeled sections with anti-caspase 3 antibody. These experiments showed no change in cell death after ARX inactivation compared with controls (data not shown) and suggested that silencing ARX promotes the exit of progenitors from the cell cycle. To test this hypothesis, we used P-H3 antibody, a marker of mitotic cells, to examine a potential reduction of the mitotic-cell fraction in ARX-silenced progenitors (Fig. 2C,D). Three days after electroporation, ∼8.4 ± 1.5% of DsRed-labeled cells in the VZ/SVZ of controls were P-H3 positive (106 cells/1253 DsRed-positive cells). However, when ARX expression was silenced, only 1.2 ± 0.8% DsRed-labeled cells in the VZ/SVZ were P-H3 positive (9 cells/756 DsRed-positive cells). A similar result was obtained with Ki67, another marker of proliferating cells (data not shown). To analyze the identities of DsRed-labeled cells, cortical cells were dispersed 24 h after electroporation and immunolabeled using the neuronal marker Tuj1. When control shRNA construct was introduced, ∼36.9 ± 5.7% of DsRed-labeled cells expressed Tuj1 (101 cells/274 DsRed-positive cells). In contrast, when S2 shRNA was introduced, DsRed and Tuj1 double-positive cells increased to ∼64.7 ± 4.6% (270 cells/417 DsRed-positive cells). These results suggest that ARX inactivation leads progenitors to exit the cell cycle prematurely and adopt the neuronal fate.

Conversely, to test the effect of ARX overexpression on cell proliferation, we introduced an ARX-overexpressing construct together with the DsRed vector in cortical progenitor cells using in utero electroporation. Contrary to ARX inactivation, there were substantially more DsRed-positive cells lining the ventricle 3 d after electroporation with CAG-IRES-EGFP-ARX compared with control (Fig. 2E,F). To establish whether these cells were still proliferating, we labeled sections with P-H3 and Ki67 antibodies (Fig. 2G,H). Whereas Ki67 is a marker for proliferating cells from S-phase through M-phase of the cell cycle, P-H3 only labels cells that are in late G₂- or M-phase. When the control plasmid was introduced, ∼8.6 ± 2% of DsRed-labeled cells in the VZ/SVZ were P-H3 positive (68 cells/793 DsRed-positive cells), and 10.4 ± 5.1% were Ki67 positive (14 cells/135 DsRed-positive cells). On the contrary, when ARX was overexpressed, only 2 ± 0.8% of the DsRed-positive cells in the VZ/SVZ were P-H3 positive (21 cells/1036 DsRed-positive cells), whereas 65.8 ± 6.6% of the DsRed-positive cells were Ki67 positive (131 cells/199 DsRed-positive cells). These results show that most ARX-overexpressing cells remained in the cell cycle, although they were not dividing. They also suggest the possibility that ARX overexpression increases the length of the cell cycle, and particularly the S- and/or G₂ phase.

To further verify this hypothesis, embryos were electroporated with either the ARX-expressing plasmid or the GFP vector alone at E13.5, injected with IdU at E16.5, and then pulse labeled with BrdU 3 h later, before being killed 2 h later (Fig. 3A). Both IdU and BrdU are thymidine analogues, which are incorporated into DNA during the S-phase of the cell cycle. Sections were then labeled with either anti-IdU antibody, which recognizes both BrdU- and IdU-labeled cells, or with an anti-BrdU antibody that recognizes specifically BrdU-positive cells (Fig. 3A). GFP⁺/IdU⁺/BrdU⁺ cells represent electroporated cells that were in S-phase in the interval of 5 h between the IdU injection and the animals being killed, and DsRed⁺/BrdU⁺ cells that were in S-phase in the interval of 2 h before the animals were killed. Thus, this experiment allowed us to determine the length of the cell cycle (Tc) and the length of the S-phase (Ts) as explained herein. If we assume that (1) cells comprising the proliferative population are asynchronously distributed in the cell cycle, (2) all cells have similar cycle times, and (3) they are uniformly distributed in the cell cycle, we can then conclude that the number of cells in each phase of the cell cycle is proportional to the ratio of the length of that phase (Fig. 3B), which means that, if we fix the brains just after an injection (t = T₀), the proportion of cells in S-phase at that time and, thus, labeled (LI₀), is equal to Ts/Tc (Nowakowski et al., 1989) (Fig. 3C). Because only the fraction Ts/Tc of proliferating cells are labeled by one injection of IdU or BrdU, the labeling index reaches a maximum when all cells in S-phase have been labeled by a series of injections. The time it takes to reach this maximum is Tc − Ts, which is the time it takes for a cell that was just at the end of S-phase at the time of the first injection to pass through G₂, M, and G₁, and then reenter S-phase (Fig. 3B). In this manner, we obtained the graph shown in Figure 3C if we consider that all cells in the VZ are proliferating cells [which means that the growth fraction (GF) equals 1] (Nowakowski et al., 1989). From the slope of the line in this graph, we can also deduce Tc (Fig. 3C).

We thus counted the proportion of GFP-positive cells in the VZ that were BrdU⁺ or BrdU⁺/IdU⁺ (Fig. 3D,E) and recorded the values obtained on a graph (Fig. 3F). We found that, in the brains electroporated with the control vector, Tc was ∼23 h and Ts = 3.5 h in the VZ (Fig. 3E). These results are very similar to what had been described by Miyama et al. (1997), who estimated 18.4 h for Tc and 4 h for Ts in the dorsal cortical VZ at the same stage (E16). However, when ARX was overexpressed, we obtained much longer times (Tc = 135 h and Ts = 41 h), confirming that ARX overexpression increases the length of the cell cycle.

Both inactivation and overexpression of ARX affect radial migration

In the developing cortex, ARX is expressed in tangentially arranged corridors in the MZ and IZ, the paths populated by migrating GABAergic interneurons arising in the GE (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). In addition, it is localized in proliferating cells in the cortical VZ, but not in radially migrating neurons (Colombo et al., 2004; Poirier et al., 2004; Friocourt et al., 2006). Because some defects in the organization of pyramidal neurons have been reported in Arx mutant mice (Kitamura et al., 2002), we examined the effects of ARX silencing in neuronal progenitors on radial migration. We performed experiments similar to those described above, and the proportion of DsRed-positive cells was established in the VZ/SVZ, IZ and cortical plate (CP), 3 or 5 d after electroporation (Fig. 4). At day 3, ARX RNAi led to an accumulation of cells in the IZ (79.1 ± 1.4%; n = 8 embryos) and a decrease in the number of cells in the VZ/SVZ (6.2 ± 0.8%) and CP (14.7 ± 1.2%) compared with controls (VZ/SVZ: 14.7 ± 1%; IZ: 50.2 ± 1.5%; CP: 35.1 ± 1.4; n = 4 embryos) (Fig. 4A–C). Interestingly, the results obtained with the negative control S2m shRNA were somewhat different from those obtained with the CAG-IRES-EGFP empty vector (Fig. 4, compare A, G), for which approximately one-half of DsRed-positive control cells (57 ± 1.4%; n = 4 embryos) had reached the CP at day 3 (Fig. 4I). To ensure that this accumulation of cells in the IZ was not an off-target effect of our RNAi construct, we used DCX 3UTR3mhp as another negative control. Both controls showed very similar cell distribution (data not shown), suggesting that the psiStrike vector itself may have some nonspecific effect on radial cell migration. However, because the results obtained with S2 shRNA were statistically different from both controls, we consider that the effect of ARX RNAi is most likely genuine. Five days after transfection, 64.9% of ARX-inactivated cells (n = 4 embryos) had finally reached the CP, suggesting that ARX suppression delayed rather than fully inhibited radial migration (data not shown).

To rule out the possibility that the observed defect in radial migration was a nonspecific effect of S2 shRNA, we used the S1 RNAi construct targeting another region of ARX (Fig. 4D–F). Although there was no major difference between the overall distribution of ARX-inactivated cells (VZ/SVZ: 11.2 ± 0.6%; IZ: 51.8 ± 1%; CP: 38.1 ± 1; n = 4 embryos) compared with control S1m (VZ/SVZ: 12.1 ± 0.8%; IZ: 51.4 ± 1.2%; CP: 36.5 ± 1.2; n = 4 embryos), we noted an accumulation of cells in the deeper aspect of the CP 3 d after electroporation (Fig. 4D,E). Thus, we examined cell distribution in the two conditions by dividing the cortex into 10 equally spaced divisions (bin 1 corresponding to the VZ of the cortex and bin 10 to the MZ) (Fig. 4D), and estimated the percentage of labeled cells in each bin. These results confirmed the accumulation of cells in the deeper part of the CP when ARX expression was silenced (Fig. 4F, bins 6 and 7). This finding strongly suggests that ARX inactivation in cortical progenitors has a direct effect on radial migration during cortical development.

Because ARX inactivation in progenitor cells seems to impair radial migration, we also studied the effect of ectopic ARX expression in radially migrating cells. Three days after transfection, only 38 ± 1.9% of ARX-expressing cells had reached the CP (n = 5 embryos), and 48.8 ± 2% were still in the IZ, compared with 32.6 ± 1.3% (n = 4 embryos) for the vector alone (Fig. 4G–I). By day 5, the majority of control cells had migrated to the CP (76.9 ± 1.4%; n = 3 embryos) compared with only 54.9 ± 1.7% of ARX-expressing cells (n = 4 embryos) (Fig. 4J–L). We also noted that the distribution of ARX-expressing cells was impaired not only in dorsal cortex, but also in lateral cortex (supplemental Fig. 2, available at www.jneurosci.org as supplemental material). Together, these results suggest that ARX expression is necessary for proper radial migration of pyramidal neurons during cortical development.

Morphology of radially migrating cells after inactivation or overexpression of ARX in cortical progenitors

To better understand the observed defect in radial migration, we examined the morphology of migrating cells. When ARX expression was knocked down using S2 shRNA, cells accumulated in the IZ (Figs. 4A–C, 5A,B). Although there was also some accumulation in the control, the morphology of the cells was different between S2 and the control shRNA (S2m or DCX 3UTR3mhp). The majority of control cells in the SVZ and the middle and lower parts of the IZ exhibited the previously described multipolar morphology (Fig. 5C, arrows and insets), whereas cells in the upper part of the IZ and in the CP had a bipolar form (Tabata and Nakajima, 2003). In contrast, ARX-silenced cells in the SVZ and IZ appeared more round, with very few or no visible processes (Fig. 5B,D). Only a few cells in the upper IZ had a normal bipolar morphology as they migrated toward the CP (Fig. 5D). These results suggest that loss of ARX expression in cortical progenitors leads to an alteration in the morphology of radially migrating neurons, resulting in their accumulation in the SVZ/IZ and impairment of their migration into the CP.

Because ectopic expression of ARX in migrating neurons also impairs radial migration, we examined the morphology of ARX-overexpressing cells. We found that the brains of E16.5 mice electroporated with CAG-IRES-EGFP-ARX contained several cells that appeared to migrate tangentially in the SVZ and lower IZ, and away from the site of injection; such cells were not present in control brains (Fig. 5E–G, arrows). In addition, most of these cells exhibited complex branching, and some had very long processes (Fig. 5F,G, insets). To quantify the number of these tangentially orientated neurons, we counted cells with visible processes in the SVZ and lower IZ of injected animals and separated them into three categories according to orientation of their main process (Fig. 5H). We found that, 3 d after electroporation, most of the control cells were radially orientated (75.9 ± 4.6%; n = 3 embryos), and only a few were orientated tangentially (0.9 ± 1%), whereas ARX-overexpressing animals displayed an increased percentage of tangentially orientated cells (41.6 ± 5.1%; n = 3 embryos) and, consequently, a decrease in the proportion of radially migrating neurons (38.2 ± 5.1%) (Fig. 5H). To rule out the possibility that these tangentially orientated cells were GABAergic interneurons electroporated while in the VZ, we stained a number of sections with GABA antibody. However, none of the DsRed-positive cells expressed GABA (Fig. 5I,J), which confirms that interneurons migrating into the cortex from the subpallium are not electroporated with this technique, as previously demonstrated (Tabata and Nakajima, 2003). Similar results were obtained using anti-calbindin antibody (data not shown). Together, these results suggest that ARX may have a role in cell morphology and particularly in the formation of processes, which may explain the radial migration defects.

The level of ARX expression is important for tangential migration

Several studies have reported the expression of ARX in interneurons migrating from the MGE to the cortex during embryonic development (Kitamura et al., 2002; Poirier et al., 2004; Colombo et al., 2004; Friocourt et al., 2006). To assess whether this gene also plays a cell-autonomous role in tangential migration, we isolated E14.5 mouse telencephalic hemispheres and performed focal electroporation into the MGE (Yozu et al., 2005). As previously, S2 and control RNAi constructs were electroporated together with the DsRed vector to visualize the transfected cells (Fig. 6). After electroporation, the telencephalic hemispheres were cultured for 40 h and cut into 100-μm-thick coronal sections, and the positions of the electroporated cells were noted. In control brains, DsRed-positive cells appeared dispersed predominantly in the GE, with a few also present in the cortex (Fig. 6A). However, in ARX-inactivated brains, most of the cells were still located in the MGE and had not migrated away from the site of injection (indicated by an asterisk). Although some cells had initiated their migration, a very small number had actually entered the cortex (Fig. 6B). To quantify this defect in migration, we estimated the proportion of DsRed-positive cells that had reached the cortex. We observed that in control brains, ∼11.3 ± 1.2% cells (n = 3 embryos) had crossed the corticostriatal boundary and had entered the cortex, whereas sections taken from brains electroporated with S2 shRNA had only 3.1 ± 0.7% cells in the cortex (n = 3 embryos). These results suggest that ARX inactivation impairs tangential migration in a cell-autonomous way. Interestingly, we did not observe any difference in cell morphology between control and ARX-inactivated cells (Fig. 6E).

We then performed similar experiment to assess the effect of ARX overexpression on tangential migration. Surprisingly, the overexpression of ARX seemed to have an even stronger effect on tangential migration. Although there was dispersion from the site of injection, most of the migrating cells only reached the corticostriatal boundary without crossing it (Fig. 6C,D). The proportion of cells that had entered the cortex was estimated and was found to be even lower than with ARX RNAi [1.8 ± 0.7 for CAG-ARX (n = 6 embryos), compared with 10.7 ± 2.4 (n = 4 embryos) for the vector alone]. To establish that this effect was not a consequence of a proliferation defect, we stained a few sections with Ki67 antibody but did not observe any difference with the control, suggesting that, in contrast to the cortex, ARX does not play any role in the regulation of cell proliferation in the basal forebrain (data not shown). We then examined the morphology of migrating cells and found that most had a bipolar appearance with a very long process, different from the typical morphology of migrating interneurons seen in controls (Fig. 6E). Taken altogether, these results indicate that ARX is necessary for tangential migration of interneurons, possibly by regulating cell morphology and process formation during migration.

ARX does not control the expression of GABA in cortical interneurons

The high degree of colocalization that exists between ARX and GABA in embryonic and adult brains and the observation that ARX expression is regulated by Dlx genes, which are strong candidates for regulating the differentiation of most, if not all, telencephalic GABAergic neurons (Stühmer et al., 2002b), have led to the suggestion that this gene might be involved in the specification of the GABAergic phenotype (Cobos et al., 2005). To test this hypothesis, we examined the degree of colocalization between ARX and GABA. We first performed immunohistochemistry on E16 rat cortical sections (Fig. 7A1–A3). As previously shown, ARX antibody labels cells in the VZ of the developing cortex and in streams of migrating interneurons that express GABA. Although the vast majority of these cells expressed both markers, we noted the presence of ARX-positive cells that were GABA negative (Fig. 7A1–A3, arrows). To further verify the degree of colocalization of these two molecules, we performed double immunolabeling experiments on dissociated cell cultures prepared from E16 rat cortices and kept in culture for 3, 6, or 9 d (Fig. 7B1–B3). We noticed at the three stages that, although most of the ARX-expressing cells were GABA-positive, not all GABAergic cells expressed ARX (Fig. 7B1–B3, arrowheads). Similar experiments were performed in ventral forebrain neurons. As previously described, our ARX antibody does not label the VZ of the MGE and LGE, suggesting that ARX is only expressed in postmitotic neurons in the ventral forebrain (Friocourt et al., 2006) (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). However, to avoid any potential contamination with progenitors that might express ARX but not GABA, we prepared dissociated cultures from E16 striatum without the VZ/SVZ and left them in culture for 3, 6, or 9 d (Fig. 7C1–C3). Similarly, we observed that only a proportion of ARX-positive neurons contained GABA and vice versa (61–65% of ARX-positive cells expressed GABA and 58–72% of GABAergic cells expressed ARX in dissociated cultures from rat E16 striatum) (Fig. 7D). These results suggest that ARX is not fully associated with the expression of GABA in both cortical and subpallial neurons.

To further test whether ARX regulates GABAergic cell differentiation, rat E16 cortical and GE cell cultures were transfected with either CAG-IRES-EGFP-ARX or the GFP-vector alone, and immunolabeled with interneuron markers such as GABA or calbindin. Two days after transfection, we found that ARX overexpression did not affect the expression of these markers in cortical or GE cultures (Fig. 7E,F). Similar results were obtained after fixing the culture 4 or 6 d after transfection (data not shown). Conversely, to assess the effect of ARX silencing, GE cultures were transfected with RNAi constructs and labeled with the same markers. As previously, we did not observe any difference in GABA or calbindin expression (data not shown). Altogether, these results suggest that ARX is neither necessary nor sufficient for the expression of the neurotransmitter GABA in the forebrain.

Role of ARX in progenitor cell proliferation

Several studies have reported that progenitor cells in the cerebral cortex express ARX, which is later downregulated as cells exit the VZ (Kitamura et al., 2002; Colombo et al., 2004; Poirier et al., 2004; Friocourt et al., 2006). Here, we demonstrated that targeted inhibition of ARX in these cells caused their premature exit from the cell cycle and differentiation into neurons. From our results, it is difficult to determine whether the effect of ARX inactivation was primarily to drive progenitors prematurely out of the cell cycle or to induce their neuronal differentiation. It is, indeed, possible that once they become postmitotic, these cells adopt a neuronal fate as a default response to environmental cues. However, the finding that ARX overexpression increased the length of the cell cycle favors a role for this gene in maintaining progenitor proliferation.

Our observations are also consistent with the microcephaly (small brain) phenotype observed in Arx mutant mice and patients with XLAG (Kitamura et al., 2002; Kato et al., 2004). This suggests that the level of ARX expression is important for the control of production of cortical neurons and the regulation of cerebral cortical size, similar to what has been described for β-catenin (Chenn and Walsh, 2003; Woodhead et al., 2006). Mutations in several genes are responsible for microcephaly in humans, including ASPM (abnormal spindle in microcephaly), microcephalin, CDK5RAP2, and CenPJ (Woods, 2004). All these genes are associated with key elements of mitosis, such as the centrosome and mitotic spindles. Although the genes regulated by ARX have not yet been identified, it would be interesting to test whether it acts on one of these elements.

Role of ARX in radial migration

Earlier studies have indicated that Arx is strongly expressed in migrating GABAergic interneurons but is absent from radially migrating cells (Colombo et al., 2004; Poirier et al., 2004; Friocourt et al., 2006). This raised the question as to what causes the misplacement of pyramidal neurons observed in the cortex of Arx knock-out mice (Kitamura et al., 2002). To establish whether ARX has a direct role in radial migration, we studied the effects of its inactivation or overexpression by using in utero electroporation. In both cases, we observed defects in neuronal migration, with cells accumulating in the IZ. We also noticed that ARX-inactivated cell bodies were oval or round in shape, with very few or no processes, very different from the multipolar morphology normally exhibited when cells exit the VZ and enter the lower IZ (Tabata and Nakajima, 2003). Since the first description of the multipolar stage during radial migration, several studies have suggested that the transition into and out of this stage is particularly vulnerable and that it is disrupted in several disorders of neocortical development, including lissencephaly (LoTurco and Bai, 2006). Cell morphology defects, very similar to those induced by RNAi-mediated inactivation of ARX, have been reported after Filamin A (Nagano et al., 2004), Rac1, or Cdk5/p27 (Kawauchi et al., 2003, 2006) inactivation. Interestingly, all these proteins interact with the actin cytoskeleton, which suggests that ARX could play a previously unsuspected role in cell morphology through actin regulation.

It is difficult to determine from our findings whether neurons fail to undergo appropriate radial migration as a consequence of the observed proliferation defect or whether neurons require a factor whose expression is controlled by ARX for their radial migration. It is important to note that, because ARX is not normally expressed in radially migrating neurons, it is not the absence of ARX itself that accounts for the migration defect. The observed changes in morphology after gain or loss of function tend to support two distinct roles for ARX. Neurogenesis and neuronal migration have in some cases appeared as two independent processes, as is the case in mice lacking the cell cycle inhibitors p19Ink4d and p27kip1, in which a population of cortical neurons fail to exit the cell cycle, but migrate to the cortex (Zindy et al., 1999). It should also be pointed out that we did not observe any change in the overall orientation of radial glial processes or their morphology (data not shown), which suggests that the neuronal migration defects are distinct from the proliferation ones.

Role of ARX in tangential migration

A number of studies have documented the absence of interneurons in the cortex of XLAG patients (Bonneau et al., 2002; Forman et al., 2005) and a defect in tangential migration of cortical interneurons in Arx mutant mice (Kitamura et al., 2002; Colombo et al., 2007), suggesting the importance of ARX in this process. To analyze the tangential migration of GABAergic interneurons from the ventral telencephalon to the developing neocortex, we used a technique that combines focal electroporation of the MGE and whole-hemisphere cultures (Yozu et al., 2005). In these experiments, we observed that both inactivation and overexpression of ARX resulted in impairment of cortical interneuron migration from the MGE. We were unable to assess whether this defect was attributable to a delay in migration rather than cessation of cell movement. The fact that there was reduced cell dispersion from the site of injection, especially for ARX-silenced cells, suggests that the motility of neurons was affected. When we examined cell morphology, we noted that, unlike control-electroporated cells, many neurons that overexpressed ARX had one unusually long process. However, we did not detect any difference in cell morphology between ARX-inactivated cells and control cells. Interestingly, a recent study found that interneurons derived from Arx mutant mice have longer processes and increased branching while migrating in a Matrigel matrix (Colombo et al., 2007). This discrepancy may be attributable to differences in experimental methods applied (RNAi vs genetic deletion). Indeed, because our shRNAs are not 100% effective, some amount of ARX protein is still present in cells targeted with RNAi.

Role of ARX in cell differentiation

The expression of ARX in regions rich in GABAergic cells as well as the diminution of cortical interneurons in patients with XLAG have prompted speculation that this gene may be involved in the specification of the GABAergic phenotype of cortical interneurons. However, we present here several arguments against this hypothesis: (1) the colocalization of ARX and GABA in cortex or GE is not complete; (2) ARX overexpression in cortical progenitors in vivo does not induce GABA or calbindin expression, even 5 d after electroporation; (3) ARX overexpression does not induce GABA expression in dissociated cultures from E16 rat cortex or GE. In addition, the observation that the introduction of an ARX-overexpressing construct into the VZ of the dorsal telencephalon induces tangential migration within the IZ, although these cells do not express GABA, suggests that ARX may be involved in migration rather than cell specification or differentiation. We thus present a model that suggests a way the ARX and Dlx genes regulate the specification and migration of cortical GABAergic interneurons (Fig. 8). Although ARX is downstream of the Dlx genes, it may only regulate migration, whereas the Dlx genes may also control the expression of GABA through other genes (Anderson et al., 1997, 1999; Stühmer et al., 2002a).

However, we cannot exclude a potential role for ARX in the specification of distinct subsets of GABAergic neurons in the subpallium. Indeed, two different studies have reported a decrease in cholinergic neurons in the basal forebrain of Arx mutants, suggesting that ARX, uniquely or in combination with other transcription factors, may play a role in the specification of at least one subpopulation of GABAergic cells (Kitamura et al., 2002; Colombo et al., 2007). In addition, the observation that ARX is still expressed in GABAergic cells in the adult brain suggests that it may also have a role in more mature neurons.

Pathophysiology of phenotypes resulting from ARX mutations

Work to date has shown that ∼10 different syndromes, with or without brain malformations, are associated with mutations of ARX. Several studies have shown that there is a connection between altered neuronal positioning and susceptibility to epilepsy (Kriegstein, 2005). Moreover, subtle alterations in interneuron position and/or function have been reported to contribute to disorders such as dyslexia, schizophrenia, or mental retardation (Kamiya et al., 2005; Galaburda et al., 2006). Our data are consistent with these findings, because both ARX inactivation and overexpression affect cell proliferation and neuronal migration, suggesting that a precise balance in the expression of this gene is necessary for the correct development of the cortex.

In the future, a better understanding of ARX-related disorders will require the identification of the pathways involving this gene. Because it is implicated in several processes that are crucial for cortical development, it is likely that some of the genes regulated by ARX may be potential candidates for neurological diseases such as epilepsy, mental retardation, schizophrenia, dyslexia, autism, and cortical malformations (such as lissencephaly and microcephaly or macrocephaly). Interestingly, a recent study has shown that mutant mice for N-cofilin, an F-actin-depolymerizing factor, display impaired radial and tangential migration, as well as an increased cell cycle exit of neuronal progenitors in the VZ (Bellenchi et al., 2007). These results, similar to what we have shown for ARX, demonstrate that mutations affecting regulators of the actin cytoskeleton contribute to the pathology of cortex development, and open new prospects concerning the pathways regulated by ARX.