The cell-intrinsic requirement of Sox6 for cortical interneuron development

Abstract

We describe the role of Sox6 in cortical interneuron development, from a cellular to a behavioral level. We identify Sox6 as a protein expressed continuously within MGE-derived cortical interneurons from postmitotic progenitor stages into adulthood. Both its expression pattern and null phenotype suggests that Sox6 gene function is closely linked to that of Lhx6. In both Lhx6 and Sox6 null animals, the expression of PV and SST and the position of both basket and Martinotti neurons are abnormal. We find that Sox6 functions downstream of Lhx6. Electrophysiological analysis of Sox6 mutant cortical interneurons revealed that basket cells, even when mispositioned, retain characteristic but immature fast-spiking physiological features. Our data suggest that Sox6 is not required for the specification of MGE-derived cortical interneurons. It is, however, necessary for their normal positioning and maturation. As a consequence, the specific removal of Sox6 from this population results in a severe epileptic encephalopathy.

Figure 1. Sox6 is primarily expressed in postmitotic Lhx6-expressing cortical interneurons

(A) To assess if migrating cortical interneurons express Sox6, coronal telencephalic sections from Dlx5/6^Cre;RCE^EGFP/+ mice were analyzed at E14.5. (A) Sox6 (red), EGFP (green) double labeled cells were observed in the mantle of the ventral telencephalon. Many of the interneurons in the cortex with morphologies suggesting active migration express Sox6, however some Sox6 expressing cells do not express EGFP (for details see a’). The dorsal ventricular zone also expresses Sox6. (B) In contrast, ‘migrating’ cortical interneurons do not express Sox5. (C) To assess if Sox6 (red) is expressed within the MGE derived lineage, we fate mapped MGE interneurons using the Lhx6^Cre;RCE^EGFP/+ (green) line. Virtually all Lhx6 lineage cortical interneurons express Sox6 (94±6%). (D) The extent of colocalization of Sox6 (green) and Lhx6 (red) was also analyzed by antibody staining at E13.5. Consistent with our genetic fate-mapping of this population, most (if not all) Lhx6 cells are also Sox6-positive, however the relative levels of expression of these two proteins varies. Sox6, while expressed at low levels in the MGE (d’), becomes highly expressed in migrating interneurons within the mantle and the cortex (d”). (E) To test if Sox6 cells are actively proliferating, we examined whether there exists coexpression of Sox6 (green) and the proliferation marker Ki-67 (red). Within the ventral telencephalon, while a few cells were double-positive (arrowheads in e’), indicating proliferation, most of the Sox6 cells did not express Ki-67. In the cortex (e’), Sox6 colocalizes with Ki-67 exclusively in the ventricular zone (arrow in e”). a’-e” corresponds to the area outlined by the white squares in A–E respectively. N=3 for each experiment condition. Scale bar in (A) corresponds to 400µm in A–B, 40µm in C, 500µm in D–E.

Figure 2. Sox 6 is expressed in mature MGE-derived cortical interneurons

(A,B) In order to determine the percentage of cortical interneurons that express Sox6 at P21, telencephalic coronal sections from Dlx5/6^Cre;RCE^EGFP/+ mice were analyzed. (A)Within the somatosensory cortex 62±3% of the total number of interneurons (EGFP) express Sox6 (red). (B) A subset of hippocampal interneurons also express Sox6 (Box in panel B, shows a higher-power view of the region indicated). (C–F) To determine the percentage of each interneuron subtype that expresses Sox6, coronal sections of somatosensory cortex were analyzed at P21. (C–E) Representative examples of double immunostaining for Sox6 (red), and SST, PV or VIP (green). (C,D) The majority of PV and SST cells co-express Sox6. (E) We observed no colocalization between Sox6 (red) and VIP (green). (F) Shows the percentage of fate mapped Dlx5/6, RCE interneurons that express Sox6 (62±3%), and the percentages of specific cortical interneuron subtypes that co-express Sox6 and the following markers: PV (93±4%); SST (94±6%); NPY (40±4%); and CR (19±2%). In contrast, VIP interneurons do not express Sox6 (2±1%). N=3 Scale bar in (A) corresponds to 40µm in A, 60µm in B, 50µm in C–E.

Figure 3. Sox 6 mutant MGE-derived interneurons have migratory defects resulting in them being largely restricted to the most superficial and deep cortical layers

(A,D) In order to determine if Sox6 affects interneuron migration, we fate-mapped (A,C,E,G) Sox6 control (Sox6^F/+;Lhx6^Cre;RCE^EGFP/+), and (B,D,F,H) mutant interneurons (Sox6^F/F;Lhx6^Cre;RCE^EGFP/+) during embryonic development (A–D) and at P1 (E–H). The specific cortical laminae were visualized using Ctip2 (which is expressed in at high levels in layer V and weakly in layer VIa at perinatal ages; Arlotta et al., 2005; Chen et al., 2008) and Tbr1 (which is expressed in layer VIa at perinatal ages; Hevner et al., 2001). While we did not detect a difference between control and mutant at E14.5 (A,B) or E15.5 (C,D); there was a noticeable difference in the distribution of Sox6 mutant interneurons by P1 (E–H). Sox6 mutant interneurons accumulate in layer VI at the expense of the more superficial layers. This abnormal distribution persists at later times, with mutant Sox6 interneurons preferentially occupying the superficial and deep cortical layers (layer I and VI, respectively), while being abnormally depleted in the intermediate cortical layers II–V (E–L). (E) The percentage of EGFP-expressing neurons in controls (black bars) and mutants (grey bars) in a given layer over the total number of EGFP in all layers at P17. 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%). (F) Total number of EGFP cells per optical field. Layer I: control (0.7±0.6) vs. mutant (22±7), Layer II/III: control (30±6) vs. mutant (18±4), Layer IV: control (24±4) vs. mutant (7±3), Layer V: control (49±9) vs. mutant (29±5), and Layer VI: control (43±11) vs. mutant (49±9); and total (I–VI): control (158±15) vs. mutant (130±10). We analyzed three controls and three mutants at each of the timepoints examined: E14.5; E15.5 and P1. For the P17 analysis, we quantified the numbers of EGFP-expressing cells in the somatosensory corticies of fours pairs of controls/ mutants. Arrow bars in (A–B) indicates blood vessels, which are labeled by the Lhx6^Cre driver (Fogarty et al., 2007). Scale bar in (A) corresponds to 200 µm in A–D and 150µm in E–H.

Figure 4. Effects of loss of Sox6 gene function on cortical interneuron marker expression

(A,B) Most of the Sox6 mutant interneurons (Sox6^F/F;Lhx6^Cre;RCE^EGFP/+) lose PV expression, however a small percentage retain low levels of PV-expression, some of which become ectopically positioned in layer I. (C,D) By contrast, Sox6 mutant interneurons upregulate NPY. (E) In the control brains (Sox6^F/+;Lhx6^Cre;RCE^EGFP/+) PV cells are distributed in layers II–VI (and absent in layer I), and a very low percentage of PV cells (green) co-express NPY (red) (4±2%). (F) In contrast, in mutant brains (Sox6^F/F;Lhx6^Cre;RCE^EGFP/+) some of the small amount of cells that keep low expression of PV are ectopically located in layer I. Virtually all the mutant PV expressing cells (green) also express NPY (red) (93±6%). E and F correspond to the squares in C and D, respectively. (G,H) Gad67 expression was slightly decreased (19±11%). (I,J) To determine if the upregulation of NPY is confined to the interneuron population we performed NPY immunostaining (red) in Dlx5/6 fate mapped interneurons (pan-cortical interneuron marker) in both (I) control (Sox6^F/+;Dlx5/6^Cre;RCE^EGFP/+), and (H) mutant (Sox6^F/F;Dlx5/6^Cre;RCE^EGFP/+) animals. We verified that the NPY upregulation in the mutant (J) is confined to the interneuron population (virtually all NPY expressing cells double label for EGFP). Quantification of the percentage of NPY expressing Dlx5/6 fate mapped interneuron shows a vast increase of NPY expressing interneurons in the mutant (72±7%) (H); vs. in the control (23±5%). The percentage was calculated as a total number of NPY-EGFP expressing cells per total number of EGFP expressing cells. We used a total number of animals of five for (A–F), and three for (G–J). Countings were performed in the somatosensory cortex at P17–P19. Scale bar in (A) 70µm in (A–D), 8 µm (E–F) and 50 µm in (G–J).

Figure 5. The cell autonomous and non-autonomous affects resulting from the loss of Sox6 gene function on subtype-specific cortical interneuron marker expression

(A–H) To determine which markers are affected in Sox6 mutant cells, we compared control (Sox6^F/+;Lhx6^Cre;RCE^EGFP/+) and mutant (Sox6^F/F;Lhx6^Cre;RCE^EGFP/+) animals at P17–P19. (A–F) Shows the percentage of fate mapped cells expressing a given marker relative to the total number of EGFP-positive neurons within that layer (marker-positive/EGFP-positive). The markers used were (A) parvalbumin (PV), (B) somatostatin (SST), (C) NPY, (D) calretinin (CR), (E) the voltage-sensitive potassium channel Kv3.1b, (F) the voltage-sensitive potassium channel Kv3.2. (G) Shows a summary of A–F showing the percentage of each marker examined, as a percentage of the total EGFP-positive cells within the somatosensory cortex (layer I–VI). Note that while the markers PV, SST, CR, Kv3.1b and Kv3.2 are decreased, the marker NPY is increased. The extent to which individual markers are altered in the mutant differs in accordance with the layer examined (see A–F). (H) To determine the non-autonomous affects resulting from the loss of Sox6 in non-MGE populations (i.e. those that are normally Sox6-negative) cortical interneurons, we compared control (Sox6^F/+;Lhx6^Cre;RCE^EGFP/+) and mutant (Sox6^F/F;Lhx6^Cre;RCE^EGFP/+) mice for the expression of the analyzed markers within the EGFP-negative population. Values were calculated by counting the total number of marker, EGFP-negative cells per optical section. Note that the only marker affected in this analysis was NPY, which was significantly increased. All countings were performed at P17–P19 and bars correspond to stand error of the mean (N=5).

Figure 6. Sox6 is genetically downstream of Lhx6

To test the genetic relationship between Sox6 and Lhx6, we both analyzed the expression of Sox6 in Lhx6 mutants (A–E), as well as the expression of Lhx6 in Sox6 mutants (F–N). Sox6 expression is dramatically decreased in Lhx6 mutant mice (Lhx6^−/−) at both E15.5 (A,B) and P15 (C,D). (A) In E15.5 control mice (Lhx6^+/−, Gad6^EGFP) migrating cortical interneurons (green) express Sox6 (red), and this expression is dramatically decreased in mutant (Lhx6^−/−, Gad6^EGFP) mice (B). By contrast, Sox6 cortical VZ expression is not affected in the Lhx6 mutant. (C,D) A decrease of Sox6 in Lhx6 mutant interneurons is still observed at P15. In order to test the extent of the Sox6 decrease in Lhx6 mutant interneurons, we fate mapped MGE-derived interneurons by examining both (C) control (Lhx6^+/−;Nkx2-1^Cre;R26R^YFP/+), and (D) mutant (Lhx6^−/−;Nkx2-1^Cre;R26R^YFP/+) alleles with the Nkx2-1^Cre driver. There was an obvious decrease in the expression of Sox6 in Nkx2-1 fate mapped Lhx6 mutant cells, while there was no change in Sox6 expression in Olig2-expressing non-neural cells. (E) Shows the percentage of Sox6-expressing interneurons in fate mapped Nkx2-1-lineage interneurons, as calculated as the ratio between the number of cells that are Sox6 positive/YFP double-positive as a percentage of the total number of YFP fate mapped cells, in either Lhx6 control (72±2%) in Lhx6^+/−;Nkx2-1^Cre;R26RYFP/+) or mutant (10±2%) in Lhx6^−/−;Nkx2-1^Cre;R26R^{YFP /+}) mice. (F–N) Lhx6 expression is not affected in Sox6 mutant cells. (F–I) We saw no difference between E14.5 controls (Sox6^F/+;Dlx5/6^Cre;RCE^EGFP/+) and mutants (Sox6^F/F;Dlx5/6^Cre;RCE^EGFP/+) in Lhx6/Lhx6 expression as assessed by in situ (F–G) and antibody staining (H,I). Similarly, we saw no difference in the expression of Sox5 in control versus mutant mice (J,K). (L,N) Lhx6 expression was still largely unaltered at P17. (N) Lhx6 expression was calculated as the total number of Lhx6-expressing cells at P17 in both the control (85±4%) in Sox6^F/+;Dlx5/6^Cre;RCE^EGFP/+) and mutant (78±6%) in Sox6^F/F;Dlx5/6^Cre;RCE^EGFP/+) mice. Scale bar in (A) corresponds to 300µm in A and B. Scale bar in (C) corresponds to 50µm in C and D, 25µm in F–J and 40 µm in L and M.

Figure 7. Physiological characterization of ectopic fast-spiking cells in layer I

A) Non-overlapping MGE-derived populations in layer I express SST and Kv3.1b. B. Post-hoc immunostaining for Kv3.1b in a recorded SST-negative fast-spiking basket cell. C. Neurolucida reconstruction of a recorded cell reveals a relative mature morphology and that the ectopic cells retain the typical innervation of cell somas (in grey). D–F Intrinsic properties associated with maturation are abnormal in Sox6 mutants. (D) Representative traces from the 500 ms protocol used from layer I mutant cells compared to a layer II/III control fast-spiking cell. (E) Significantly lower F_max (p=0.047), higher ISI adaptation (p=0.0004) and larger Sag (p=0.020) suggest a less mature phenotype. F. The mutant cells fails to maintain a high frequency firing during a prolonged 5s protocol. (G) The frequency of EPSPs is similar in Sox6 mutants and control littermates. Abbreviations: SST, somatostatin.

Figure 8. EEG characterization

A) Mutant P16 EEG recordings in cortical fields (M1) reveal dysrhythmic theta oscillations interrupted by high amplitude delta waves (asterisk) often associated with hippocampal epileptic discharges in CA1 (arrows). B) Interrictal epileptic activity becomes more prominent during slow wave sleep with independent discharges in the cortex (arrow) and hippocampi (asterisk). C) Although usually asymptomatic and considered interictal, simultaneous epileptic discharges in the hippocampi and polyspike complexes in the cortex occasionally manifest as generalized myoclonus. D) Generalized seizures with synchronous bi-frontal onset often remain confined to cortical fields, with variable hippocampal involvement. (Acquisition: 2000Hz, HP 0.1 Hz, LP 300 Hz, filtered at LP 70Hz for display)

Figure 9. Spectral Analysis

Spectral analysis (FFT: consecutive 1 sec segments, averaged over six 10 sec epochs) during slow wave sleep at P17 (±0.5D) reveal spectral band amplitude (sq root power) modifications with increased delta power band and decreased theta power band in cortical EEG (A+C) reflecting frequent high amplitude delta waves (B, asterisk). In addition, delta, beta and gamma powers were increased in hippocampal recordings (A arrows: additional peaks at 25.5±.5Hz and minor peak at 53±3Hz, Averaged power increase: C) reflecting frequent epileptic bursts composed of high amplitude delta waves with superimposed polyspikes (B, arrow).