Maturation-Promoting Activity of SATB1 in MGE-Derived Cortical Interneurons

Summary The generation of cortical interneuron subtypes is controlled by genetic programs that are activated in the ventral forebrain and unfold during the prolonged period of inhibitory neuron development. The LIM-homeodomain protein LHX6 is critical for the development of all cortical interneurons originating in the medial ganglionic eminence, but the molecular mechanisms that operate downstream of LHX6 to control the terminal differentiation of somatostatin- and parvalbumin-expressing interneurons within the cortex remain unknown. Here, we provide evidence that the nuclear matrix and genome organizer protein SATB1 is induced by neuronal activity and functions downstream of Lhx6 to control the transition of tangentially migrating immature interneurons into the terminally differentiated Somatostatin (SST)-expressing subtype. Our experiments provide a molecular framework for understanding the genetic and epigenetic mechanisms by which specified but immature cortical interneurons acquire the subtype-defining molecular and morphophysiological characteristics that allow them to integrate and function within cortical circuits.


INTRODUCTION
Motor planning and execution, sensory representation, and cognitive activity depend on the integrated function of cortical circuits that consist of glutamatergic projection neurons and GABAergic local circuit interneurons. In rodents, cortical interneurons constitute approximately 20% of all cortical neurons and are extremely diverse (Markram et al., 2004). They are generated during embryogenesis in two transient subpallial structures, the medial and caudal ganglionic eminence (MGE and CGE), and reach the cortex by tangential migration (Wonders and Anderson, 2006;Welagen and Anderson, 2011). Although the different groups of interneurons are endowed with subtype-specifying genetic programs at their sites of origin in the ventral forebrain, during tangential migration they are morphologically indistinguishable and lack many of the properties of functionally mature inhibitory neurons Cossart, 2011). Termination of tangential migration and entry into the cortical plate heralds the onset of terminal differentiation and maturation, which culminates in the generation of distinct interneuron subtypes identified by characteristic morphological, electrophysiological and molecular properties (Butt et al., 2005). For example, the neuropeptide somatostatin (SST) and the calcium-binding protein parvalbumin (PV) mark nonoverlapping groups that originate in the MGE, while the vasoactive intestinal peptide (VIP) is expressed specifically by CGE-derived interneurons (Kawaguchi and Kubota, 1997;Markram et al., 2004;Rudy et al., 2011).
We and others have previously demonstrated that the LIM homeodomain (HD) transcription factor LHX6 plays a pivotal role in the generation of MGE-derived cortical interneurons. Lhx6 is induced early in all MGE-derived interneuron precursors and is required in a dose-dependent manner for the differentiation of the SST + and PV + groups of interneurons and the assembly of functional inhibitory circuits Zhao et al., 2008;Neves et al., 2012). Although Lhx6 controls the development of all MGE-derived interneurons, the unfolding of Lhx6dependent differentiation and maturation programs follows subtype-specific spatiotemporal patterns. This is underscored by the differential spatiotemporal expression profile of subtype markers, such as PV and SST. Both Pv mRNA and protein are expressed only at relatively late stages of interneuron development in the postnatal cortex (del Río et al., 1994;Taniguchi et al., 2011). In contrast, Sst mRNA is expressed during embryogenesis by tangenitally migrating immature interneurons, although the SST peptide is detected exclusively in postnatal animals (Taniguchi et al., 2011;Neves et al., 2012). These observations suggest that region-and stage-specific extrinsic factors are likely to interact with intrinsic genetic determinants to regulate the different phases of cortical interneuron development in the mammalian forebrain. The identity of such extrinsic signals and the intrinsic factors that regulate interneuron differentiation downstream of LHX6 remain largely unknown.
In this study, we identify the genome organizer Special AT-rich DNA Binding Protein 1 (SATB1) as a key regulator of cortical interneuron development. We provide evidence that Satb1 is specifically activated in MGE-derived interneurons within the cortical plate in an activity-dependent manner. By manipulating its expression in the MGE, we demonstrate that SATB1 inhibits the tangential migration and controls the differentiation of SST + interneurons downstream of LHX6. In addition, genetic ablation of Satb1 leads to differentiation deficits primarily of the SST + group of cortical interneurons in vivo. Together, our studies identify SATB1 as a critical regulator of interneuron maturation and terminal differentiation in the mammalian cortex.

Satb1 Is Expressed in MGE-Derived Cortical Interneurons following Entry into the Cortical Plate
To identify genes implicated in the development of cortical inhibitory circuits, we carried out a microarray-based comparative profiling of gene expression of dorsal forebrain from embryonic day (E) 15.5 wild-type and Lhx6-null mice  (see Experimental Procedures). Our screen identified genes already known to be expressed in cortical interneurons in an also referred to as Kv3.1,Npas1,Npy,Sst,Sox6) Zhao et al., 2008; and others that have been implicated in the differentiation of diverse cell types, but their role in the development of cortical interneurons is currently unknown. Among the genes of the latter group was Satb1, which encodes a HD nuclear matrix protein that controls T cell differentiation and cancer metastasis via chromatin organization and epigenomic modifications Cai et al., 2003Cai et al., , 2006Han et al., 2008). The complete set of genes affected by deletion of Lhx6 in the dorsal forebrain is shown in Table S1.
To determine the developmental profile of Satb1 expression in cortical interneurons, brain sections from E12.5-P2 Gad67 GFP mice were double immunostained for SATB1 and GFP. SATB1 + interneurons were not detected in the dorsal forebrain of E12.5 embryos and were infrequent in the cortex of E15.5 embryos (Figure 2A; data not shown). At these stages, Satb1 was not expressed in the MGE or in tangentially migrating interneurons (Figures 2A-2C2). In contrast, a large number of GFP + SATB1 + interneurons was detected within the CP and MZ of E18.5 Gad67 GFP embryos ( Figures 2D-2D2). At later stages (P2), SATB1 + interneurons were absent from the MZ and IZ/SVZ but were abundant within the cortex ( Figures 2E-2F2). Finally, and in agreement with previous reports (Huang et al., 2011;Balamotis et al., 2012), Satb1 was also expressed by pyramidal neurons . By demonstrating that during cortical development Satb1 is expressed by an increasing number of MGEderived GABAergic neurons, our results suggest that SATB1 is implicated in the terminal differentiation and maturation of cortical interneurons.

Ectopic Expression of SATB1 in the MGE Induces a Gene Expression Profile Characteristic of Mature Cortical Interneurons
To determine whether SATB1 promotes the maturation of cortical interneuron precursors, we conducted a series of misexpression experiments in the MGE of E14.5 embryos and analyzed changes in expression of selected marker genes by qPCR (Figures 3A and 3B and Extended Experimental Procedures). As expected (Figure 2), SATB1 was undetectable in control MGE, but electroporation of a pCAGGs-based SATB1encoding expression vector (pSATB1-RFP) led to high-level expression of nuclear-localized SATB1 protein (Figures S1B-S1C2). Twenty-four hours after electroporation, the level of mRNAs encoding RAC1, a small Rho GTPase with multiple roles in the development of inhibitory neurons in the forebrain (Chen et al., 2007) and KCNC1, a pottasium channel that marks PV + interneurons (Chow et al., 1999), remained unchanged, while transcritps for PV and CR were undetectable in both control and Satb1-expressing MGEs ( Figure 3B and data not shown). In contrast, transcripts encoding DCLK2, which is transiently detected in tangentially migrating interneuron precursors (our observations and Tuy et al., 2008), were downregulated (Figure 3B). In addition, ERBB4, a tyrosine kinase receptor widely expressed by tangentially migrating interneurons (Flames et al., 2004) but restricted postnatally mainly to PV + interneurons (Fazzari et al., 2010;Neddens and Buonanno, 2010), and NKCC1, a Na-K-Cl cotransporter expressed in immature GABAergic interneurons (Yamada et al., 2004;Inada et al., 2011), were also downregulated ( Figure 3B). Conversely, mRNAs for KCC2, a K + /Cl À exchanger expressed in mature interneurons and the overlapping subtype markers SST and NPY, were significantly upregulated ( Figure 3B). The upregulation of Kcc2 by SATB1 in MGE is likely to reflect an in vivo regulatory scenario since all KCC2 + interneurons in the cortex of E18.5 GAD67 GFP embryos coexpressed SATB1 ( Figures 3C-3D3). The SATB1-induced downregulation of genes associated with tangentially migrating immature cortical interneurons (Dclk2,ErbB4,and Nkcc1) and the concomitant upregulation of Kcc2 and the subtype markers SST and NPY in MGE precursors support the idea that SATB1 drives the maturation of the SST + group of cortical interneurons.

SATB1 Promotes the Maturation of SST + Interneurons
To examine whether misexpression of Satb1 in immature interneuron precursors leads to SST production, a feature of mature interneurons of the postnatal cortex, dissociated E14.5 MGEs transfected with pSATB1-RFP were plated onto feeder layers of neonatal mouse cortex and analyzed by immunostaining after 2, 7, and 14 days in vitro (DIV; Figure 4A). Expression of SATB1 increased dramatically the percentage of MGE cells that produced SST at all stages analyzed ( Figures 4B-4D). Interestingly, the maximal increase in the percentage of RFP + SST + cells was evident by 2DIV ( Figure 4D), suggesting that upregulation of Sst mRNA (by 24 hr; Figure 3B) and SST protein in this assay represent immediate early effects of Satb1 misexpression in interneuron precursors. SATB1 also led to a significant increase in the fraction of transfected cells that coexpress NPY, a neuropeptide expressed by a subset of SST + interneurons (Fogarty et al., 2007) (at 2DIV, pRFP: 7.2% ± 2.1% versus pSATB1- RFP: 29.3% ± 1.4%, p < 0.01, n = 3 independent electroporations). In contrast to the upregulation of SST and NPY, no PV + cells were detected during the 2 week period we examined (data not shown). Taken together, our results suggest that SATB1 is sufficient to induce in immature MGE precursors a molecular phenotype that is normally encountered in mature SST + cortical interneurons.
To examine the requirement of Satb1 activity for SST induction, we adopted a short-hairpin (sh)RNA approach to knock down endogenous Satb1 in interneuron precursors ( Figure S1D). pGFP-V-RS (control), pGFP-V-RS-scrShRNA (scrambled small hairpin RNA [ShRNA] control) and pGFP-V-RS-Satb1ShRNA constructs were transfected into E14.5 MGEs, which were subsequently dissociated and cultured for 7 days on cortical feeder layers ( Figure 4A). Immunostaining for GFP (to identify transfected cells) and SST showed that expression of the Satb1 ShRNA resulted in a dramatic decrease in the percentage of SST + cells relative to controls ( Figure 4E). These experiments argue that Satb1 activity is required in our coculture system for the differentiation of MGE precursors into SST + interneurons.
To explore the effect of Satb1 on the differentiation of interneurons in vivo, E14.5 MGEs transfected with control or pSATB1-RFP vectors were mechanically dissociated and the resulting cell suspension was grafted into the cortex of P0 mice ( Figure  S2A) (Xu et al., 2010). One month later, the transplanted cells were identified in the cortex by RFP expression and their subtype identity was analyzed. Consistent with previous reports (Miyoshi et al., 2007), the majority of MGE cells transfected with the control vector dispersed throughout the cortex and differentiated predominantly into PV + and (to a lesser extent) SST + interneurons (65% and 20%, respectively; Figures S2B-S2B2, S2D-S2D2, and S2F). In contrast, fewer pSATB1-RFP-expressing MGE cells were identified within the cortex, suggesting that premature overexpression of Satb1 in MGE precursors inhibits their migration and dispersion (see below). Interestingly though, we observed a significant increase in the representation of SST + cells (33.1% ± 0.4%) and a corresponding decrease of PV + cells (9.27% ± 3.3%) within the population of grafted MGE cells that expressed Satb1 within the cortical layers ( Figures S2C-S2C2, S2E-S2E2, and S2F). Although this analysis is confounded by the apparent effect of SATB1 overexpression on the migration of grafted MGE cells, it reinforces the idea that SATB1 promotes the maturation of interneurons into the SST + subtype. Together, these experiments demonstrate that overexpression of SATB1 in MGE precursors induces a neuropeptide phenotype that characterizes mature cortical interneurons of the SST + group.

Overexpression of SATB1 Blocks Tangential Migration of Interneuron Precursors
Maturation of interneurons is associated with termination of tangential migration (Bortone and Polleux, 2009). To examine whether SATB1 is sufficient to promote the sessile state of inter-neuron precursors, pSATB1-RFP was electroporated into the MGE and its effect on the tangential migration of interneuron precursors was analyzed by culturing forebrain slices for 48 hr ( Figure S3A). As expected, MGE cells expressing a control vector showed robust tangential migration toward the cortex ( Figures S3B and S3B1). Moreover, the majority of these cells were characterized by the typical morphology of tangentially migrating interneurons, highlighted by the presence of a long leading process (arrow in inset of Figure S3B1; Bellion et al., 2005). In all cases, MGE cells expressing Satb1 failed to migrate tangentially and were localized exclusively at the site of electroporation in the MGE ( Figures S3C-S3E). We conclude that SATB1 inhibits the tangential migration and promotes the sessile state of cortical interneurons.

Satb1 Activity Is Required In Vivo for the Differentiation of SST + Cortical Interneurons
To examine whether SATB1 is required for the development of cortical interneurons in vivo, we analyzed mice homozygous for a knockout allele of Satb1 (Satb1 tm1a(EUCOMM)Hmgu ; called hereafter Satb1 EUCOMM ) (for details on the generation and analysis of the phenotype of Satb1 EUCOMM mice see Experimental Procedures and Supplemental Information). No reduction in the total number of cortical interneurons (as assessed by in situ hybridization histochemistry for Gad1) was observed in different cortical areas of P15 Satb1 EUCOMM homozygous mice ( Figures  5A, 5B, 5G, and S4A). In addition, the number of LHX6 + interneurons in the motor, somatosensory (bregma À1.94 mm) and visual cortex of Satb1 mutants was normal, although a modest ($18%) reduction of these cells was observed in the somatosensory cortex (S1BF; bregma 0.38 mm) of Satb1deficient animals ( Figures 5C, 5D, 5G, and S4A). In contrast, we observed a dramatic reduction in the number of neurons expressing Sst mRNA ($63%) and SST protein ($62%) in all cortical areas of P15 Satb1 mutants (Figures 5E-5I, 5L, and S4A). In addition, we observed a small decrease ($14%) in the number of PV + interneurons in all cortical areas of Satb1 mutants we examined ( Figures 5J-5L). Finally, no significant difference was observed in the number or distribution of CGEderived VIP-expressing interneurons ( Figures S4B and S4C) between control (99.7 ± 20.6) and Satb1-deficient animals (87.7 ± 9.3, n = 3 animals per genotype). These results argue that Satb1 activity is required in vivo primarily for the development of the SST + group of cortical interneurons.
To examine whether deletion of Satb1 affects selectively expression of Sst, we analyzed additional markers, which are expressed by subsets of SST + interneurons. Among them are the neuropeptides corticotropin releasing hormone (CRH) and NPY and the proteins CR and nNOS (Wonders and Anderson, 2006;Kubota et al., 2011). We observed a significant decrease in the number of CR + and NPY + interneurons in the S1BF area of Satb1 mutants ( Figures 5M-5Q). We also observed a consistent trend toward reduced numbers of CRH + interneurons when all cortical layers of S1BF were analyzed ( Figures 5R-5T). However, a clear reduction of CRH + interneurons was documented in the bottom (V-VI) cortical layers ( Figure 5T), in which interneurons coexpressing CRH and SST are located (Kubota et al., 2011). Finally, no difference was observed in the number  (G) Quantification of Gad1-, Lhx6-, and Sst-expressing cells in the somatosensory cortex (S1BF, bregma 0.38 mm) of wild-type (white bars) and Satb1-deficient mice (black bars). (H-K) Immunofluorescence of P15 cortical sections from wild-type (H and J) and Satb1-deficient (I and K) mice with antibodies specific for SST (H and I) and PV (J and K). (L) Quantification of SST + and PV + cells in the somatosensory cortex (S1BF, bregma 0.38 mm) of wild-type (white bars) and Satb1-deficient mice (black bars). (M-P) immunofluorescence of P15 cortical sections from wild-type (M and O) and Satb1-deficient (N and P) mice with antibodies specific for CR (M and N) or NPY (O and P). (Q) Percentages of CR + and NPY + cells in wild-type (white bars) and Satb1-deficient cortex (black bars). of type I nNOS + projection interneurons (Tomioka et al., 2005;Perrenoud et al., 2012) in the cortex of control (22.6 ± 0.6 per surface area) versus Satb1-deficient mice (19.1 ± 3.2 per surface area, n = 3 animals per genotype) (Figures S4D and S4E). These findings indicate that the effect of Satb1 deletion is not restricted to the expression of Sst arguing that the gene controls multiple aspects of the maturation program of SST + , interneurons.
Interestingly, despite the dramatic reduction of both Sst mRNA and protein in the cortex of Satb1 EUCOMM mice, the number and distribution of tangentially migrating Sst(mRNA) + interneurons during embryogenesis were indistinguishable between control and mutants ( Figures S5A-S5D). In addition, no difference in the expression of Lhx6 was observed in tangentially migrating interneurons or in the cortex of E18.5 wild-type and Satb1 mutant embryos ( Figures S5E and S5F; data not shown). Since expression of Lhx6 and Sst mRNA are early markers of MGE-derived interneuron precursors Neves et al., 2012), these experiments suggest that Satb1 activity is dispensable for the early specification of the SST group of interneurons and is required for their maturation within the pallium.
To further explore this hypothesis, we analyzed the expression of KCC2, a nonspecific marker of maturation of multiple groups of interneurons and pyramidal neurons, in the cortex of control and Satb1 mutant embryos. At E18.5, we observed a dramatic decrease in the expression of KCC2 in mutant cortex ( Figures  S5G and S5H). Since at this stage the majority of KCC2 + cells are interneurons (Figure 3), these experiments provide further evidence that Satb1 activity is required in vivo for the maturation of cortical interneurons. The expression of Satb1 in principal cortical neurons raises the possibility that the cortical interneuron defects observed in Satb1 mutants are secondary to deficits in cortical lamination. However, this is unlikely as we failed to detect any changes in cortical lamination in the cortex of Satb1 EUCOMM homozygous mice using in situ hybridization for the layer-specific markers ER81 and CUX2 (data not shown; see also Liodis et al., 2007). Taken together, our experiments indicate that Satb1 activity is required in vivo for the maturation of the SST + group of MGE-derived cortical interneurons.

Lhx6-Dependent Differentiation of SST + Cortical
Interneurons Requires Satb1 Activity Satb1 was identified in our genome-wide microarray screen as a gene downregulated in Lhx6-deficient mouse brain, suggesting that its expression depends on Lhx6 activity. To investigate the epistatic relationship between Satb1 and Lhx6, we analyzed the levels of SATB1 in the cortex of Lhx6 mutant mice  that were also transgenic for the Nkx2.1 CRE ; Rosa26 EYFP reporter. Consistent with our gene expression profiling, we found that, in the brain of P15 Lhx6 À/À mice, the proportion of YFP + cells expressing Satb1 was dramatically reduced relative to that of control mice (Figures 6A-6C). These experiments indicate that Lhx6 activity is necessary for Satb1 expression in MGE-derived cortical interneurons.
A common feature of Lhx6 Zhao et al., 2008) and Satb1 (this study) mutant mice is the differentiation deficit of MGE-derived cortical interneurons. To further examine whether LHX6 and SATB1 function in a common cascade, we overexpressed combinations of control (RFPonly), pSATB1-RFP and pLHX6-RFP expression vectors in the MGE of E14.5 mouse embryos and immunostained for SST following 7 days coculture with P0 cortical feeder layers (Figure 4A). Expression of pLHX6-RFP alone resulted in an $2fold increase in the percentage of RFP + cells coexpressing SST, while expression of pSATB1-RFP led to an $6-fold increase in the percentage of SST + RFP + interneurons (Figure 6D). Interestingly, cotransfection of SATB1-and LHX6encoding vectors did not result in a further increase in the percentage of SST + RFP + cells beyond the levels achieved by expression of SATB1 alone ( Figure 6D). These findings are consistent with our phenotypic analysis of Satb1 mutants and support the idea that LHX6 and SATB1 are components of a common developmental cascade.
The Satb1-dependent upregulation of SST in dissociated MGE cultures (Figure 4) allowed us to test whether Satb1 activity mediates the effect of LHX6 on SST induction. For this, LHX6 was overexpressed in E14.5 MGEs along with control (GFP-scrShRNA) or Satb1-specific (GFP-Satb1ShRNA) ShRNA constructs, and SST expression was analyzed following dissociation and culture of transfected cells on cortical feeder layers for 7 days. Coelectroporation of pGFP-V-RS-Satb1ShRNA and pLHX6-GFP vectors resulted in a dramatic reduction in the fraction of SST + cells relative to the pLHX6-GFP and pGFP-V-RS-scrShRNA combination ( Figure 6E), suggesting that Satb1 is required for the Lhx6-mediated upregulation of SST and differentiation of MGE progenitors.
To test whether SATB1 is sufficient to rescue SST expression in Lhx6-deficient interneuron precursors, pSATB1-RFP was introduced into Lhx6-deficient MGEs that were subsequently cultured for 7 days. Relative to control vectors, expression of SATB1 increased dramatically the percentage of MGE cells that were positive for SST ( Figure 6F), indicating that SATB1 is sufficient to drive expression of Sst downstream of LHX6. Nevertheless, the efficiency of SST induction in mutant MGE cells was considerably smaller relative to that observed in their wild-type counterparts ( Figure 6D), arguing that additional factors missing from Lhx6 mutant MGE cells are implicated in the control of SST expression.

LHX6 Is Not Sufficient to Drive Expression of Satb1
During development, Satb1 is induced in MGE-derived cortical interneurons several days after the expression of Lhx6 (Figure 2), suggesting that LHX6, even though necessary (Figure 6), is not sufficient to induce expression of Satb1. Consistent with (R and S) In situ hybridization histochemistry of P15 cortical sections from wild-type (R) and Satb1 mutant (S) mice with a Crh-specific riboprobe. (T) Quantification of CRH + neurons in the layers of the somatosensory cortex (S1BF, bregma 0.38 mm) of wild-type (white bars) and Satb1-deficient mice (black bars). n = 3 animals per each interneuron marker analysis, *p < 0.05, **p < 0.01, ***p < 0.001 for each test (Student's homoscedastic t test with two-tailed distibution). See also Figures S4, S5, and S7. this idea, overexpression of LHX6 in MGE precursors (by electroporating pLHX6-GFP) did not upregulate Satb1 (data not shown). Moreover, in utero coelectroporation of pLHX6-GFP and a MASH1-expressing plasmid into cortical projection neuron progenitors, which is sufficient to upregulate the early interneuron marker DCLK2, failed to induce expression of Satb1 ( Figures S6A-S6F2). To further explore the mechanisms that regulate expression of Satb1, we tested the idea that KCC2, which is coexpressed in mature cortical interneurons with SATB1, is an upstream regulator of Satb1. However, electroporation of the Kcc2-encoding pKcc2-GFP vector ( Figures  S6G-S6I2)

Activity-Mediated Induction of Satb1
Spontaneous neuronal activity, a feature of the developing CNS, influences fundamental processes of brain development, such as cell migration, neuronal maturation, and synaptogenesis (Moody and Bosma, 2005). In the mouse neocortex, spontaneous activity is observed from around E16.5 and peaks at early postnatal stages (Corlew et al., 2004), raising the possibility that neuronal excitability controls the upregulation of SATB1 observed at perinatal stages. To explore this possibility, we established cultures of dissociated dorsal telencephalon from E14.5 mouse embryos and analyzed the expression of Satb1 in GABAergic interneurons at various intervals after plating ( Figure 7A). At 24 hr, only a small fraction of interneurons were positive for SATB1 but the percentage of SATB1 + GAD67 + cells increased considerably at subsequent stages ( Figures 7B-7E and 7N), reflecting the developmental profile of SATB1 in endogenous cortical interneurons. Interestingly, treatment of 24 hr cultures with KCl (40 mM), which is capable of depolarizing immature neurons, led to a significant upregulation of c-FOS and SATB1 within 24 hr (Figures 7F-7K and 7O). KCl-induced depolarization can trigger Ca 2+ influx through voltage-gated calcium channels, a subtype of which (L-type) is known to be expressed in cortical interneurons and have been implicated in several activity-dependent developmental processes in the brain (Jiang and Swann, 2005). Consistent with this, KCl treatment of cortical cultures in the presence of the Ca 2+ channel blocker nifedipine (10 mM) abrogated the KCl-mediated induction of SATB1 ( Figures 7K,  7L, and 7O).
In immature cortical interneurons, GABA has an excitatory effect due to a reverse chloride gradient and several studies suggest that this neurotransmitter influences neuronal migration, maturation, and integration into circuits (Ben-Ari, 2002;Ben-Ari and Spitzer, 2010). To explore whether GABA is part of the activity stimulus that upregulates Satb1 in our cortical cultures, we treated 24 hr cortical cultures with two independent GABA(A) receptor (GABAAR) antagonists, bicuculine (10 mM) and picrotoxin (10 mM). Both resulted in a relatively small but statistically significant reduction in the percentage of SATB1 + GAD67 + neurons at 48 hr ( Figures 7M and 7P). Taken together, these studies suggest that neuronal excitability and Ca 2+ activity are implicated in the upregulation of Satb1 in cortical interneurons and that this upregulation is partially mediated by GABA receptors. In (C), n = 3 animals; in (D)-(F), n = 3 independent electroporations for each experimental condition, **p < 0.01, ***p < 0.001 for each test (Student's homoscedastic t test with two-tailed distibution). See also Figures S1 and S6.

DISCUSSION
Development of cortical interneurons in mammals is a prolonged process that is initiated during embryogenesis in the ventral telencephalon and completed in juvenile animals with the functional integration of highly divergent GABAergic neurons into cortical circuits . The astonishing diversity of cortical interneurons is ultimately determined by genetic programs activated in the ventral forebrain but the full spectrum of molecular, morphological and physiological properties of mature interneurons becomes apparent only at relatively late stages of interneuron development and coincides with the onset of neuronal activity (Butt et al., 2005;Miyoshi et al., 2010;Cossart, 2011). Considerable recent progress has identified genetic cascades that control the specification of cortical interneurons, but the molecular mechanisms that underlie the maturation of different subtypes remain largely unknown . Here, we demonstrate that the chromatin organizer and transcription factor SATB1 has a pivotal role in the maturation and terminal differentiation of a major group of MGE-derived cortical interneurons and provide evidence that spontaneous neuronal activity is a critical regulator of Satb1 expression in the pallium. Our combined loss-and gain-of-function studies demonstrate that SATB1 coordinately controls the expression of subtypespecific and interneuron-wide genes in a manner that characterizes mature interneurons of the Sst group. Based on these findings, we argue that SATB1 links subtype-specific genetic programs that are activated in the ventral forebrain and control the specification of SST + interneurons with molecular cascades that promote the terminal differentiation and maturation of all classes of inhibitory neurons within the pallium.
Consistent with the observation that Satb1 is specifically expressed in interneurons within the cortical plate, a Satb1-null mutation in mice resulted in a dramatic downregulation of Sst mRNA and protein in cortical interneurons of postnatal animals but had no effect on Sst expression in their tangentially migrating precursors. These findings argue for a two-stage regulation of Sst expression: an early SATB1-independent phase, which denotes subtype commitment of SST + interneurons, and a subsequent SATB1-dependent phase, which reflects the maturation of SST-specified MGE precursors into functional interneurons. This biphasic mode of regulation of Sst is consistent with the complex regulatory landscape of the locus (Burbach, 2002) and suggests that distinct stage-specific transcriptional regulators control the expression of Sst during the course of cortical interneuron development. A recent report has shown that in mouse brain SATB1 occupies an upstream regulatory region of Sst (Balamotis et al., 2012), suggesting direct transcriptional regulation of this locus by SATB1. Although this finding provides a pertinent explanation for our current observations, the detailed mechanisms by which SATB1 controls the coordinate expression of interneuron-specific genes requires further investigation. During T cell development and in breast cancer cells, it is thought that target loci of SATB1 are tethered via specialized genomic sequences to a common regulatory framework that recruits chromatin-modifying factors and allows the simultaneous response to transcriptional complexes Cai et al., 2003;Han et al., 2008). These studies and our current findings suggest that SATB1 functions as a global genome organizer to coordinately control the expression of multiple loci that promote maturation and integration of SST + interneurons into functional cortical circuits.
SST is detectable exclusively in cortical interneurons of postnatal animals suggesting that in mammals productive expression of Sst is associated with the later SATB1-dependent phase of expression. This is supported by our demonstration that overexpression of SATB1 in MGE precursors is sufficient to induce expression of both Sst mRNA and protein. These findings argue that the absence of SST from the MGE and tangentially migrating interneurons is not due to the nonspecific inhibition of Sst mRNA translation in these cells and raise the intriguing possibility that, in addition to its transcriptional role, SATB1 is implicated in the posttranscriptional regulation of Sst expression in cortical interneurons. Although the mechanisms of such regulation are currently unclear, it has been previously reported that nNOS1 and PSD95 are posttranscriptionally regulated in pyramidal neurons in an activity-dependent manner by the product of the fragile X syndrome-associated FMR1 locus (Todd et al., 2003;Kwan et al., 2012). It appears therefore that in the mammalian cortex posttranscriptional gene regulation is a feature of both excitatory and inhibitory neurons and that SATB1 could be a key mediator of activity-dependent posttranscriptional regulation in cortical interneurons.
Although both major groups of MGE-derived interneurons express Satb1, our analysis of Satb1-deficient mice revealed a relatively modest deficit of PV + inhibitory neurons. Such deficit could reflect a secondary effect of the developmental abnormalities of SST + interneurons observed in these mutants, an idea which is supported by the emerging view that abnormal maturation of specific subsets of GABAergic interneurons in the cortex often lead to generalized defects of other subpopulations of interneurons across the entire neuronal network (Cossart, 2011). The additional dendritic and synaptic transmission deficits of principal cortical neurons observed in Satb1 mutants (Balamotis et al., 2012) could also contribute to the PV + interneuron deficit. Alternatively, the reduced number of PV + interneurons in Satb1-deficient animals could result from a cell autonomous requirement of this factor for the differentiation of a subset of PV + interneurons. Subtype-specific deletion of Satb1 in vivo should distinguish between these possibilities.
Previous studies from our and other groups have demonstrated that the LIM HD transcription factor LHX6 is an upstream regulator of divergent molecular cascades that control the development of MGE-derived cortical interneurons Zhao et al., 2008). Downstream of LHX6, the transcriptional regulator SOX6 controls predominantly the differentiation of PV + interneurons (Azim et al., 2009;. Here, we provide evidence that expression of Satb1 and the consequent effect on the development of SST + interneurons depend on Lhx6 activity. The considerable delay of Satb1 activation relative to Lhx6 in vivo and our overexpression studies indicate that LHX6 is not sufficient to drive expression of Satb1 and suggest that additional stage-specific regulators may be involved. The demonstration that neuronal excitability is sufficient to upregulate Satb1 expression in cultured interneurons provides a frame-work for understanding the mechanisms by which LHX6 controls later stages of interneuron development. We hypothesize that LHX6 is required cell autonomously in cortical interneurons for the advent of a gene expression profile that allows these cells to develop and/or respond to the emerging activity of the developing brain. Previous studies have indicated that ambient GABA plays a key role in the development of early activity of the brain (Ben-Ari et al., 2007), raising the possibility that GABA-mediated activity controls the expression of Satb1 in an LHX6-dependent manner in cortical interneurons. In support of this hypothesis, we have previously reported dramatic changes in the release of GABA by inhibitory neurons in the hippocampus of Lhx6-null mutant mice (Neves et al., 2012). Moreover, our current pharmacological experiments suggest a requirement for GABAAR for the activation of Satb1 in cortical interneuron cultures. Nevertheless, the observed inhibition of Satb1 expression in these experiments was only partial, suggesting that additional mechanisms may be implicated in the induction of this gene. In summary, our experiments argue that SATB1 constitutes an activity-dependent molecular switch downstream of Lhx6, which controls the maturation and terminal differentiation of cortical interneurons. These findings have important implications for understanding the molecular control of cortical interneuron development and inhibitory network assembly.
Additional methods and any associated references are available in the Extended Experimental Procedures.

EXTENDED EXPERIMENTAL PROCEDURES Animals
Two independently generated ES cell clones with the Satb1 EUCOMM were transmitted through the germline to establish Satb1 EUCOMM heterozygous mice. Targeting of the Satb1 locus was validated by Southern blot and PCR analysis of genomic DNA from ES cells and heterozygous animals (data not shown). Immunostaining of brain sections from Satb1 EUCOMM/EUCOMM mice with a SATB1-specific antibody confirmed deletion of Satb1 in homozygous mice ( Figure S7). Similar to the phenotype previously reported for an independently generated null mutant strain , Satb1 EUCOMM homozygous mice were born but failed to thrive and died within the first three weeks.

Gene Expression Profiling of Dorsal Forebrain from Lhx6-Null Mutant Mice
Dorsal forebrain from E15.5 embryos resulting from intercrossing Lhx6 +/À mice was dissected and stored in RNAlater (QIAGEN). Total RNA was isolated using RNeasy (QIAGEN) according to the manufacturer's protocol. Microarray experiments and bioinformatics analysis was performed by Oryzon (Oryzon Genomics, Parc Cient'fic de Barcelona, Spain). RNA samples were labeled with specific fluorochromes and hybridized to cRNA controls on the DNA microarrays. The microarray images were acquired using an Agilent scanner and the raw data were processed with proprietary Oryzon software. Bioinformatics analysis was applied to the results using the proprietary Oryzon tool Polyphemous TM , which normalizes the raw data, conducts an analysis of the copies, defines the list of genes that undergo a significant change in the level of gene expression, and defines the ontology and the metabolic or regulatory pathways that are involved. From that point, data mining was used to carry out our biological analysis.

Quantification of MGE Transcripts
For quantification of transcripts following electroporation with pSatb1-RFP, the area of MGE expressing the highest levels of RFP was dissected 24 hr after transfection, and RFP + cells were isolated by flowcytometry (FACS ARIAII, Becton Dickinson; typically a yield of 10000-20000 cells). Total RNA was purified (RNeasy microKit, QIAGEN) and subjected to reverse transcription (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems) and quantitative PCR according to manufacturer's instructions (Taqman Gene Expression Assays, Applied Biosystems). Each independent experiment represents a triplicate of qPCRs.

Expression Constructs
Full length cDNAs for Satb1 (IMAGE clone ID: 4164993), Kcc2 (IMAGE clone ID: 6838880) and Lhx6  were cloned into modified pCAGGs-based expression vectors. The pCAGGS-IRES-GFP was a gift from James Briscoe (NIMR) and pCAGGS-IRES-RFP was a gift from Francois Guillemot (NIMR). Expression was confirmed by Western blot analysis or immunostaining of transfected P19 embryonal carcinoma cells or brain slices ( Figure S3 and data not shown). Satb1-specific shRNA expression pGFP-V-RS vectors were obtained from Origene (TG501973) and their efficiency to downregulate expression of Satb1 was tested by Western blot analysis in P19 cells transfected with pSatb1-RFP ( Figure S1D).

Electroporations, MGE Cell Culture. and Transplantation
Ex vivo electroporation of MGE in embryonic brain slices was conducted as described previously (Stü hmer et al., 2002). Twelve hours after electroporation, MGE regions with the strongest RFP or GFP signal were dissociated as previously described (Du et al., 2008). The resulting cell suspension was either cocultured with feeder cells prepared from neonatal cortex (P0-P1) (Xu et al., 2004) or grafted into neonatal (P0-P1) cortex as described (Xu et al., 2010). Transplanted animals were transcardially perfused at P30, and dissected brains were processed for immunohistochemistry. In utero electroporations were carried out as previously described (Nguyen et al., 2006).

P19 Cells and Western Blotting
Mouse embryonal carcinoma P19 cells were grown in DMEM supplemented with 10% FCS, 2 mM glutamin and 1% penicillin/streptomycin (all from GIBCO) and transfected with lipofectamin according to the manufacturer's protocol (Lipofectamin â 2000 reagent, Invitrogen). Cells were then mechanically homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors (all from SIGMA) and total protein extractions were obtained. The proteins were separated on 4%-12% gradient gels using the XCell Surelock Mini-Cell and NuPAGE â MES SDS Running Buffer (Invitrogen) and transferred to nitrocellulose membranes (Amersham) using the XCell II Blot module and NuPAGE â Transfer Buffer (Invitrogen). Membranes were blocked with Blotto A (Santa-Cruz) and incubated O/N with primary antibodies for Satb1 (goat polyclonal anti-Satb1, Santa-Cruz; 1:1000) and actin (Sigma; 1:1000) diluted in the blocking buffer. The next day after washing in TBST, membranes were incubated for 1 hr at RT with HRP-conjugated secondary antibodies (Santa-Cruz; 1:2000). The immunoreactive bands were observed with ECL according to manufacturer's instructions (Pierce â ECL Western Blotting Substrate, ThermoScientific).

Immunostaining
For immunostaining on brain sections from mice P15 or older, animals were transcardially perfused with 4% PFA and brains were postfixed overnight (O/N). Vibratome (70 mm) or cryostat (14 mm) sections were permeabilized with PBT (0.5% Triton X-100 in Figure S4. Analysis of Interneuron Markers in P15 Wild-Type and Satb1-Deficient Mice, Additional Results Related to Figure 5 (A) Quantification of GAD1 + , LHX6 + and SST + interneurons in the motor (M1, bregma 0.38 mm), somatosensory (S1, bregma À1.94 mm) and visual (V1, bregma À2.92 mm) cortex of wild-type (white bars) and Satb1-deficient (black bars) mice. The number of Sst mRNA-expressing interneurons is significantly reduced in all areas examined. (B-E) Immunofluoresence on P15 cortical sections from wild-type (Satb1 +/+ ; B and D) and Satb1-deficient (Satb1 À/À ; C and E) mice with antibodies specific for VIP (B and C) and nNOS and SST (D and E). n = 3 animals per genotype, *p < 0.05, **p < 0.01, ***p < 0.001 (Student's homoscedastic t test with two-tailed distribution). Figure S5. SATB1 Is Not Required for the Specification or Tangential Migration of Cortical Interneurons, Additional Results Related to Figure 5 (A-D) In situ hybridization histochemistry on forebrain brain sections from E14.5 (A and B) or cortical sections from E18,5 (C and D) wild-type (A and C) or Satb1 À/À (B and D) mouse embryos using an Sst-specific riboprobe. (E and F) Immunostaining of cortical sections from E18.5 wild-type (E and G) and Satb1-deficient (F and H) mice, immunostained for LHX6 (E and F) or KCC2 (G and H) and counterstained with DAPI. Although there is no difference in the number of LHX6 + cells at this stage between the two genotypes, note the dramatic reduction of KCC2-specific signal in Satb1-deficient relative to control embryos.