Migrating cortical interneurons express Sox6
We identified Sox6 in a previous microarray analysis to be expressed in cortical interneurons (Batista-Brito et al., 2008
). In order to analyze the expression of Sox6, we used the pan-interneuron transgenic line Dlx5/6Cre;RCEEGFP/+
, which allows for the permanent labeling of interneurons with EGFP through Cre-mediated recombination of the RCE reporter. Immunocytochemistry of Sox6 demonstrated that migrating cortical interneurons express this protein at all of the analyzed time points (E12.5, E13.5: data not shown; E14.5: ). Furthermore, Sox6
is also expressed in other cortical populations, particularly within the ventricular zone (VZ) of the dorsal telencephalon ().
Sox6 is primarily expressed in postmitotic Lhx6-expressing cortical interneurons
Due to the high degree of similarity between Sox6
(Lefebvre et al., 2007
; Lai et al., 2008
), we tested if Sox5 was expressed in migrating cortical interneurons (). While Sox5 is expressed in postmitotic pyramidal cells, it is excluded from cortical interneurons. Indeed, our analysis revealed that Sox5 and Sox6 expression is complementary within the neocortex (c.f. ).
To determine whether Sox6 is expressed within a specific subpopulation of cortical interneurons, we used genetic fate-mapping and immunocytochemical co-localization to examine its overlap with Lhx6, a marker of MGE-derived cortical interneuronal lineages (Cobos et al., 2006
; Du et al., 2008
; Fogarty et al., 2007
; Liodis et al., 2007
), (). Sox6 and Lhx6 are extensively co-localized within the MGE, with the vast majority of Lhx6 expressing cells also being Sox6 positive (: 94±6% based on the colocalization of EGFP and Sox6 in Lhx6cre RCEEGFP/+
mice). However, while Sox6 is highly expressed in post-mitotic migrating interneurons, its level of expression is lower within the MGE (), in contrast to Lhx6 whose levels are similar in both proliferative and migrating interneurons ().
To determine if Sox6 expressing cells are mitotic, we examined the expression of the proliferation marker Ki-67 (Kill, 1996
) (). In the ventral telencephalon only a small percentage of Sox6 cells were undergoing proliferation within the SVZ (). In contrast, Sox6 expressing cells within the cortical VZ uniformly express Ki-67, suggesting that Sox6 is uniformly expressed in all the progenitors giving rise to pyramidal cell lineages ().
Sox6 is expressed in mature MGE-derived cortical interneurons
By fate mapping cortical interneurons using the pan-interneuron transgenic labeling strategy Dlx5/6Cre;RCEEGFP/+, we determined that 62±3% (N=3) of interneurons within the P21 cortex express Sox6 (). We also detected extensive Sox6 labeling in hippocampal interneurons (). While the vast majority of Sox6 cells in the cortex are interneurons, most of the remaining ones express Olig2 (data not shown).
Sox 6 is expressed in mature MGE-derived cortical interneurons
In the murine somatosensory cortex, parvalbumin (PV), somatostatin (SST) and vasoactive intestinal polypeptide (VIP) are expressed in mutually exclusive populations of cortical interneurons (Kubota et al., 1994
; Miyoshi et al., 2007a
). By genetic fate mapping it has recently been demonstrated that PV- and SST-expressing interneurons are exclusively derived from the MGE, while VIP cells do not come from that lineage (Fogarty et al., 2007
; Xu et al., 2004
). By contrast, CR and NPY, label interneuron populations derive from both the MGE and the CGE (Butt et al., 2005
; Fogarty et al., 2007
; Xu et al., 2004
). Hence, we performed co-immunohistochemistry for Sox6 and these interneuron markers (, N=3). This analysis revealed that the vast majority of PV- (93±4%) and SST-expressing (94±6%) interneurons are Sox6 positive, while virtually all the VIP interneurons are Sox6 negative (2±1%). Furthermore, we found that 40±4% of NPY, and 19±2% of CR interneurons co-express Sox6. Taken together, these results demonstrate that Sox6 is expressed in most mature MGE-derived interneurons.
Sox6 loss of function
null mice are generally born alive, but the majority of them die within an hour of birth from unknown causes (Smits et al., 2001
). A very small percentage of Sox6
null mice live up to P10–11; however, these mice are consistently smaller and weaker than their control littermates. To test the functional role of Sox6
in cortical interneurons, we began by analyzing the surviving Sox6
null mice (Sox6−/−
) at P10–P11. While the total number of cortical interneurons, as assessed by Gad67
expression was not obviously severely affected (Supplemental Figure 1A,B
), the interneuron markers PV and SST appeared to be greatly decreased, while the numbers of NPY expressing cells appeared to be dramatically increased (results not shown). In contrast, VIP-expression was not affected in the Sox6
mutants. These findings are consistent with a similar analysis examining the role of Sox6
in these same contexts (Azim et al., 2009
The most striking of these findings was the loss of PV-expressing cells. However, given the late initiation of expression of this protein (approximately P12 in wildtype cortical basket cells) coupled with the high level of lethality in the mutant, it is difficult to know whether these observations reflect a loss of this population or simply their failure to mature in Sox6
mutant mice. Hence, we sought to examine the effect of conditional ablation of Sox6
specifically within the MGE-derived cortical interneuron populations. By using the Lhx6Cre
driver in combination with a Sox6F/F
conditionally-null allele we were able to restrict the removal of Sox6
solely to the MGE-derived population (Fogarty et al., 2007
). The intersection of Lhx6
- and Sox6
-expression allows for exquisite specificity for testing the function of Sox6
within cortical interneurons derived from the MGE. By including the RCEEGFP/+
reporter line in our analysis, we were able to fate map the Lhx6/Sox6
expressing cells in both controls (Sox6F/+;Lhx6Cre;RCREGFP/+
) and conditional mutants (Sox6F/F;Lhx6Cre;RCEEGFP/+
). Moreover, by examining the EGFP-negative cortical interneuron population, we could gauge the non-cell autonomous consequences of loss of Sox6
on CGE lineages. Sox6F/F;Lhx6Cre;RCEEGFP/+
animals generally resembled their non-mutant littermates up to approximately P15. However, they subsequently failed to thrive and developed a severe seizure disorder, ultimately resulting in death between the ages of P17–P19.
Sox6 is necessary for cortical interneuron migration and laminar positioning
mutant cells did not show any obvious defect in tangential migration (c.f with Lavdas et al., 1999
; Marin and Rubenstein, 2001
). Upon reaching the cortex, interneurons shift to a radial mode of migration as they enter the cortical plate (Ang et al., 2003
; Polleux et al., 2002
). Our data appear to indicate that conditionally null Sox6
MGE-derived interneurons have a defect in their ability to transition from tangential to radial migration. Consequently they accumulate in the marginal (MZ) and intermediate (IZ) zone. To provide landmarks for specific cortical laminae, we used the perinatally-expressed transcription factors Ctip2 (: which is expressed in layer V and at lower levels in layer IVa; Arlotta et al., 2005
; Chen et al., 2008
) and Tbr1 (: which is expressed in layer VIa; Hevner et al., 2001
). Postnatally, mutant Sox6
) accumulate in layers I and VI, apparently at the expense of layers II, III and IV (; : Layer I: control (0.5±0.3%) vs. mutant (17±6%), Layer II/III: control (20±3%) vs. mutant (15±3%), Layer IV: control (16±3%) vs. mutant (5±3%), Layer V: control (31±4%) vs. mutant (22±5%), and Layer VI: control (28±4%) vs. mutant (36±8%)). This population is normally absent from layer I as shown in control littermates (; ). Indeed, normally the only cortical interneurons in this layer are CGE-derived (GF unpublished data) and a small population of NPY interneuron derived from the preoptic area (Gelman et al., 2009
). Hence, at P17–P19 although redistributed () the total number of fate-mapped interneurons is only slightly decreased (18%±12% decrease; ). The mutant cells do however appear to retain their GABAergic interneuron character, as their expression of Gad67
persists. However there we did observe a reduction in this population comparable to the observed decrease in fate-mapped neurons (19%±11%, ).
Sox 6 mutant MGE-derived interneurons have migratory defects resulting in them being largely restricted to the most superficial and deep cortical layers
Effects of loss of Sox6 gene function on cortical interneuron marker expression
Sox6 mutant interneurons retain their identity but fail to mature
We next performed double immunostainings for EGFP and multiple molecular markers characteristically expressed by different cortical interneuron subtypes (,, N=5). The most dramatic effects observed in the mutant population were a marked decrease in PV expression (by 94±3%; , ) and a concomitant increase in NPY expression (by 77±6%; , ). There was also a 30±2% reduction in the SST-expressing fate-mapped population and more specifically a complete loss of the SST/CR double-positive subtype (). The percentage of fate-mapped interneurons expressing the molecular markers Kv3.1b (normally expressed in PV-fast spiking cells at P17) and Kv3.2 (normally expressed in PV-fast spiking cells and some SST-expressing cells) was also accordingly decreased (). Furthermore, the levels of these markers in those neurons in which expression persisted were markedly reduced (). To ascertain the degree of cell death in conditional Sox6 mutants compared to control animals we performed caspase 3 cleavage staining at E13.5, P1 and P17. At no age did we observe an obvious difference in apoptosis (results not shown). Consistent with this result, we observed that 93±6% of the weakly PV-expressing cells displaced population in layer I were NPY positive (compared to 4±2% in control animals).
The cell autonomous and non-autonomous affects resulting from the loss of Sox6 gene function on subtype-specific cortical interneuron marker expression
We also examined if there was any change in the number or location of non-MGE-derived interneurons (i.e. Lhx6-negative). This was accomplished in both control and conditional Sox6 mutant animals by determining the number and location of EGFP-negative, VIP-expressing and CR-expressing (SST-negative) interneurons. We observed no overall alteration in these interneuron subtypes in the absence of Sox6 ().
NPY is upregulated both autonomously and non-autonomously in conditional Sox6 null mutants
We observed that 23±5% of the Dlx5/6-expressing population are NPY-positive in control mice. However, when this population is examined in Sox6 conditional null mice, 72±7% of all interneurons express NPY. In addition, in both control and conditionally null mice, all NPY staining within the cortex is confined to interneurons (i.e EGFP-expressing cell, ). This observation allowed us to examine NPY expression in both MGE-derived and non-MGE-derived lineages by investigating the expression of NPY in Sox6F/F;Lhx6Cre;RCEEGFP/+ animals (; ). We reasoned that since NPY is confined to the interneuron population, all NPY/EGP-double positive cells in the cortex of these mice must be MGE-derived, while NPY-positive/EGFP-negative cells are not. NPY expression was strongly upregulated both autonomously (, 16±2% in control versus 65±4% in mutants; i.e the percentage of EGFP-expressing cells that are NPY-positive) and non-autonomously (, 30±3% in control versus 48±5% in mutants; i.e numbers of EGFP-negative/NPY-positive cells per area measured).
Sox6 functions genetically downstream of Lhx6
The co-expression of Sox6 and Lhx6 within both the MGE, as well as in the postmitotic cortical interneurons derived from this structure, is near complete. Moreover, Lhx6
null mice resemble Sox6
mutants in that MGE-derived cortical interneurons are similarly mis-positioned, die at a comparable age and are deficient in similar cortical interneuron populations (Liodis et al., 2007
; Zhao et al., 2008
). Taken together this suggests that within MGE-progenitors, there exists a genetic interaction between Lhx6
. To test this hypothesis, we analyzed whether the reciprocal loss of Lhx6
within MGE-derived lineages affects the other’s expression (). At E15.5, in contrast to control neurons, MGE-derived Lhx6
mutant interneurons do not express Sox6, as assessed through Sox6-immunostaining of mice on a Gad67EGFP
(Tamamaki et al., 2003
) background (). However, within the cortical VZ the expression of Sox6 was unperturbed ().
Sox6 is genetically downstream of Lhx6
To more specifically assess the genetic relationship between these two genes, we examined Lhx6 heterozygote (Lhx6+/−) and homozygote mutant (Lhx6−/−) interneurons on a Nkx2-1Cre;R26RYFP/+ background, which allowed us to directly visualize MGE-derived interneuron lineages independent of Lhx6 expression (). Using this approach, a similar result was obtained at both P2 (data not shown) and P15 (). In Lhx6 mutant animals, we found that the vast majority of YFP-expressing cortical interneurons lose their expression of Sox6 (an 86±4% decrease in the number of MGE fate-mapped interneurons expressing Sox6 in Lhx6−/− compared with Lhx6+/− animals). As noted above, in control animals we also observe a population of non-neural cortical cells that co-express Sox6 and Olig2 and the proportion of these double-labeled cells was not affected in Lhx6 mutants ().
In contrast, Lhx6 expression was not affected in Sox6 mutants (85±4 in controls and 78±6 in mutant animals, per optical area examined; ). At E14.5, Sox6 mutant cortical interneurons (Dlx5/6Cre fate-mapped EGFP-expressing cells) express Lhx6 mRNA () and Lhx6 protein (). Furthermore, these cells retain their Lhx6 expression at later ages (). Taken together these results indicate that in MGE-derived cortical interneurons although Lhx6 is required for Sox6 expression, Sox6 is not necessary for Lhx6 induction or maintenance. This leads us to conclude that Sox6 functions genetically downstream of Lhx6.
Sox5 does not compensate for the loss of Sox6 in cortical interneuron lineages
Given the close homology between Sox5
, we were curious to determine whether any functional compensation by Sox5 occurs within cortical interneurons upon loss of Sox6
. To address this issue specifically within the MGE-derived cortical interneuron populations, we examined whether the expression of Sox5 is cell-autonomously upregulated upon loss of Sox6
. In these animals, Sox5 maintained a normal pattern of expression, and there was no indication that this protein is upregulated within the MGE-derived cortical interneuron population ( and data not shown). This is distinct from the findings of a recent paper that shows that Sox5
is upregulated in the Sox6
null mouse (Azim et al., 2009
). Taken together, these results suggest that at least within Lhx6 lineages, there is no evidence for compensation by Sox5 in Sox6
conditional mutants and hence the upregulation of Sox5
in the absence of normal Sox6
expression is not obligate.
We were also intrigued to explore the role of Sox6
within the pallium. To specifically remove Sox6
from cortical lineages, we bred the Sox6F/F
conditional allele onto an Emx1Cre
background (N=3 for control and mutants). We investigated whether the loss of Sox6 in the pallium had an effect on P21 pyramidal neurons by examining the expression of SatB2 (Supplemental Figure 2A,B
, a marker for callosal projecting pyramidal neurons; Alcamo et al., 2008
; Britonova et al., 2008) and Ctip2 (Supplemental Figure 2C,D
, expressed in corticofugal pyramidal neurons; Arlotta et al., 2005
; Chen et al., 2008
) in these mutants. To determine if the loss of this gene has a non-autonomous effect on cortical interneurons, we also examined these mutants for the expression of PV, SST and NPY at P21 (Supplemental Figure 2E–K
). Neither analysis produced evidence that the loss of Sox6
in the cortical primordium has an effect on cortical development. In addition, we did not observe any overt behavioral phenotype or seizures in these animals (results not shown).
Sox6 mutant FS-basket cells exhibit immature intrinsic properties
The loss of Sox6 results in the ectopic positioning of Lhx6-lineage cells to the periphery of the cortical plate, and to layer I in particular. This provided us with the unique opportunity to record the electrophysiological properties of cells of known subtypes within layers where they do not normally reside. The Lhx6-lineage includes all MGE-derived cortical interneurons, and is largely comprised of two main subgroups that express SST and PV, respectively. The latter group contains a rather homogenous population of basket cell interneurons characterized as fast-spiking (FS). Although PV is lost in the majority of Sox6 mutant interneurons (, ) SST-expression is less affected. We performed whole-cell current clamp recordings of EGFP-labeled cells in layer I of acute brain slices from P14-16 mice, with subsequent post-hoc immunohistochemistry for SST and Kv3.1b. All cells (n=4) that proved to be negative for SST possessed a firing pattern that clearly resembled that of wild-type FS-interneurons. Kv3.1b expression in these cells while present was strongly reduced compared to controls (). The dendritic arbors of these cells were large and highly arborized, and the axons formed the typical “baskets” around the cell somas of neighboring cells () suggesting that they are relatively mature. In order to assay the maturity and integration of the mutant cells in greater detail, we compared their electrophysiological properties to those of FS cells in layer II/III of control animals ( and Supplementary Table 1; n=3).
Physiological characterization of ectopic fast-spiking cells in layer I
The mutant cells were indistinguishable from the control cells with regards to passive membrane properties, such as resting membrane potential (RMP), input resistance (Rin
) and membrane constant tau (See Supplementary Table 1). Similarly, the spike and afterhyperpolarization (AHP) kinetics, such as half spike width, AHP time to the lowest point and AHP amplitude were not significantly different. However, the multiple spike dynamics of the mutant cells had a lower max firing rate and a more pronounced intra spike interval (ISI) adaptation (; p=0.047 and p= 0.0004 respectively), and were unable to sustain firing during prolonged (5s) protocols (). Furthermore, mutant cells exhibited a higher firing threshold ( p=0.019) and more pronounced sag (7.1±1.5mV vs. 2.9±1.9 mV; p=0.020) during hyperpolarizing steps, collectively suggesting that these cells are retarded in their maturation (Itami et al., 2007
; Okaty et al., 2009
A characteristic of the immature cortex is that the frequency of EPSPs in FS-basket cells increases dramatically during development between P10 and P14 (Okaty et al., 2009
). To test whether the ectopically positioned FS interneuron population received normal levels of excitatory input, we held the cells at −72mV for at least 2 minutes and measured the frequency at which they received excitatory input. Perhaps surprisingly, there was no difference between the mutant ectopic cells and the control layer II/III FS-cells (). In conclusion these results suggest that with regards to the mutant cells despite having normal morphology and receiving appropriate levels of input their electrophysiological properties suggest that maturation of FS cells in impaired in these mutants.
Removal of Sox6 in MGE derived lineages leads to a severe epileptic encephalopathy
Sox6 mutants were undistinguishable from their littermates until approximately 15 days of age, at which point they develop spasticity as evidenced by an abnormal scissoring posture of the limbs when mutants were held by the tail. By P16, they became progressively more withdrawn and developed spontaneous seizures. They died between P17 and P19 of a combination of prolonged seizures and dehydration.
To further assess the seizure phenotype, four mutants (two Sox6F/F; Dlx5/6Cre and two Sox6F/F; Lhx6Cre were combined as they had indistinguishable phenotypes) and four wild-type controls were monitored daily by video-EEG from P16.
The epileptic phenotype was found to be temporally progressive in all recorded mutants. During early P16, mutants remained able to explore and presented minimal cortical EEG anomalies. They were able to generate background theta rhythms similar to controls, although these were somewhat less well sustained and repeatedly intertwined with slower delta waves (). Already at P16, the interictal trace revealed bilateral synchronous bursts of epileptic activity in the hippocampi characterised by fast polyspikes overlying high amplitude delta waves. This epileptic anomaly was accentuated during slow wave sleep () and were occasionally accompanied by cortical delta waves or polyspikes, behavioural manifested as slowing or rapid axial myoclonus (). This was followed by the development of generalised seizures () manifested by an axial myoclonus followed by subtile generalized clonic movements. Epileptic activity during these seizures was most often confined to the cortex, with intermittent recruitment of the hippocampi.
Spectral analysis performed on slow wave sleep epochs at late P16.5 and early P17 illustrated the interictal anomalies described above. Examination of the cortical EEG (M1) revealed increased spectral amplitude in the delta frequency band (228 µV/Hz in mutants vs 163 µV/Hz in controls, n=4). In addition, epileptic bursts observed in the hippocampi were reflected in spectral analyses as increased spectral amplitude in the delta frequency band (, 292 µV/Hz in mutants vs 199 µV/Hz in controls), as well as in the beta (43 µV/Hz vs 30.8 µV/Hz) and gamma (28 µV/Hz vs 17 µV/Hz) frequency bands (). Notably, the spectral amplitude estimates in CA1 revealed a significant new peak around 25±3Hz in the beta range with a minor peak around 53±3Hz in the gamma range ().