The requirement of Nkx2-1 in the temporal specification of cortical interneuron subtypes

Abstract

Previous work has demonstrated that the character of mouse cortical interneuron subtypes can be directly related to their embryonic temporal and spatial origins. The relationship between embryonic origin and the character of mature interneurons is likely reflected by the developmental expression of genes that direct cell fate. However, a thorough understanding of the early genetic events that specify subtype identity has been hampered by the perinatal lethality resulting from the loss of genes implicated in the determination of cortical interneurons. Here, we employ a conditional loss-of-function approach to demonstrate that the transcription factor Nkx2-1 is required for the proper specification of specific interneuron subtypes. Removal of this gene at distinct neurogenic time points results in a switch in the subtypes of neurons observed at more mature ages. Our strategy reveals a causal link between the embryonic genetic specification by Nkx2-1 in progenitors and the functional attributes of their neuronal progeny in the mature nervous system.

Figure 1. Temporally regulated genetic fate mapping and Nkx2-1 loss-of-function in Olig2^CreER precursors

(A) The genetic strategy for examining Nkx2-1 conditional loss-of-function. while simultaneously fate mapping of MGE-derived precursors This is achieved by combineing the Olig2^CreER driver with a floxed Nkx2-1 (Nkx2-1^C) allele for conditional loss-of-function and the Z/EG reporter line for genetic fate mapping purposes. Expression from the Z/EG reporter line is directed from the CAG, Hybrid promoter and contains a Polyadenylation signal (pA). (B) The Olig2 locus directs the expression of the inducible form of the Cre recombinase, CreER, in Nkx2-1^C/+ control and Nkx2-1^C/−conditionally mutant embryos in a pattern identical to the endogenous Olig2 transcript. (C) Administration of tamoxifen (4mg) at E10.5 and concomitant activation of Cre activity results in the complete loss of Nkx2-1 from the MGE at E12.5 in Nkx2-1^C/−conditionally mutant embryos and in the permanent labeling with EGFP of MGE-derived cells as they exit the ventricular zone of Nkx2-1^C/+ control and Nkx2-1^C/− conditionally mutant embryos. Lhx6, whose expression is directly activated by Nkx2-1 and provides a convenient postmitotic marker for MGE-derived interneuron populations, is downregulated as a consequence of the loss of Nkx2-1.

Figure 2. Conditional loss of function of Nkx2-1 at early (E10.5) and late (E12.5) time points is accompanied by changes in expression of genes involved in interneuron development

The affects on gene expression of inducible loss of Nkx2-1 function at E10.5 (A–C) and E12.5 (D) were examined two days later using in situ hybridization of coronal sections of E12.5 (A–C) and E14.5 (D) embryos. (A) Shh, Lhx6 and Lhx7, whose expression is dependent on normal levels of Nkx2-1 in the MGE, are downregulated in Nkx2-1^E10.5LOF mice. These are indicative of a reduction in the specification of the interneuron and cholinergic neuron lineages. (B) By contrast, Gad67 and Dlx2 are expressed at apparently normal levels within the subpallium, revealing that ventral GABAergic neuron development does not appear to be affected by the conditional loss of Nkx2-1 in Nkx2-1^E10.5LOF mutant mice. The histogram below this photomicrograph demonstrates that the density of GAD65-positive neurons is not significantly decreased in the mutant population compared to the wild type controls. (C) The expression of Nkx6-2, Gli1 and Coup-TFII, which normally are normally expressed in both the ventricular zone of the sulcus separating the MGE and LGE, as well as portions of the CGE, are expanded throughout most the MGE ventricular zone in Nkx2-1^E10.5LOF mice. Similarly, Islet1, which is normally confined to the SVZ and postmitotic regions of the LGE, expands such that it is also expressed through the SVZ of the MGE of in Nkx2-1^E10.5LOF mutants. (D) Treatment of conditional Nkx2-1 mice with tamoxifen at E12.5 effectively removes Nkx2-1 expression by E14.5. Expression of Shh, Lhx6 and Lhx7 is also reduced in Nkx2-1^E12.5LOF mutant mice suggesting that the loss of this gene at this age affects both the character of GABAergic and cholinergic interneuron lineages. Arrows in A, C and D indicate the position of the MGE. Error bars in the histogram of B represent SEM.

Figure 3. Both the presence of myoclonic seizure activity and a reduction of GABAergic cortical inhibition are observed in Nkx2-1^E10.5LOF mutant mice

(A–D) Electroencephalographic examination of freely moving Nkx2-1^E10.5Ctrl control (A) and Nkx2-1^E10.5LOF mutant P15 mice reveals abnormal interictal discharges (B) during normal resting behavior. The initial period of a spontaneous seizure episode is characterized by the appearance of continuous single spike activity (C) As shown in this EEG trace recorded shortly after the onset of seizure, increases in frequency and amplitude occur within minutes and progress (D) into continuous spike and spike-wave rhythmic activity. This gradually returns to normal cortical activity at the end of the seizure episode. (E–F) In situ hybridization on brains from electroencephalographic monitored mice shows a correlation exists between the presence of seizure episodic behavior and the reduction in the number of GABAergic neurons in the cortex. This reduction is due mainly to a loss of MGE derived interneurons characterized by the expression of Lhx6. Nkx2-1^E12.5LOF mutant mice display only a modest reduction of inhibitory profiles in the cortex when compared to Nkx2-1^E10.5LOF mutants (F), insufficient to disrupt the normal cortical activity observed by a lack of seizure episodes in those mice.

Figure 4. The generation of MGE-derived interneuron subtypes is altered in both Nkx2-1^E10.5LOF and Nkx2-1^E12.5LOF mutant mice

(A–C) In comparison to littermate controls, (A) the morphological and molecular profiles of cortical interneurons are altered in Nkx2-1^E10.5LOF (B) and Nkx2-1^E12.5LOF (C) mutant mice. In Nkx2-1^E10.5LOF mutants (B) the pronounced loss of EGFP-expressing PV and Sst cells is accompanied by a proportional increase in the number of VIP- and CR-expressing interneurons. Nkx2-1^E12.5LOF mutants (C) display the same shift towards VIP and CR profiles in the superficial layers (layers II, III and IV) but not in the deeper layers (layers V and VI) of cortex. Interestingly, in Nkx2-1^E12.5LOF mutants the Sst population seems unaffected. (D) In Nkx2-1^E10.5LOF mutants the shift in the overall molecular profile of EGFP-labeled interneurons is accompanied by a dramatic decrease in their number. However, even taking into account the reduction in the absolute numbers of genetically fate mapped cortical interneurons, there is a total net increase in the number of EGFP-labeled VIP and CR cells. This suggests that the loss of Nkx2-1 at this timepoint results in a fate switch in many of labeled MGE-derived neurons. This alteration in fate is even more apparent in Nkx2-1^E12.5LOF mutants. In these mice there is a 5-fold and a 2-fold absolute increase in the numbers of EGFP-expressing VIP and CR cells, respectively. Moreover, in these same mice there is only a slight overall change in the total number of cortical interneurons. (E–G) In Nkx2-1^E10.5LOF mutants where a net loss of fate mapped (EGFP+) cells destined to cortical regions is observed (E) there is a large increase in the number of EGFP-expressing neurons with morphologies, consistent with them being striatal medium spiny projections neurons. (G). These cells express DARPP-32 (F) supporting the notion tNkx2-1 is also required for the determination of the interneuron versus medium spiny projection neuron identity. Error bars in the histograms of B, C, E and H represent SEM. A single asterisk above the histogram indicates P<0.05, while two asterisks indicate P<0.01, as evaluated by a Student T-test.

Figure 5. Conditional loss of function Nkx2-1 at an early (E10.5) and later (E12.5) time point results in a shift in the electrophysiological profile of fate-mapped EGFP+ neocortical interneurons

(A–C) electrophysiological profiles and morphological reconstructions (D) of EGFP+ interneurons obtained from whole cell patch clamp recording performed on control (Nkx2-1^C/+) acute in vitro brain slices. (A) high frequency non-adapting discharge of a fast spiking (FS) interneuron in response to a supra-threshold current injection (+0.6nA, 500ms). (B) non-fast spiking (NFS) interneuron displaying slow adaptation in spike frequency in response to spike threshold current injection (+150pA) superimposed on trace exhibiting prominent voltage sag but no rebound spike in response to hyperpolaring step (−100pA). (C) intrinsic burst spiking interneuron characterized by multiple spikes on rebound (arrow) from hyperpolarizing current step (−30pA) to −80mV and adapting spike frequency discharge at spike threshold (+20pA). (D), morphological reconstruction of the IB interneurons shown in panel (C); axon, red; dendrite, blue. (E–G) Recordings and morphology (H) of EGFP+ interneurons recorded from conditional loss-of-function animals (Nkx2-1^C/−). (E) Initial adapting interneurons (iAD) exhibited pronounced adaptation in spike height and frequency when injected with supra-threshold current step (+375pA). (F) Profile of a late spiking interneuron characterized by a steady ramp depolarization in response to near spike threshold current injection (+36pA) and at threshold (+40pA) a significant delay to spike onset (arrow). (G) Slowly adapting (sAD) interneuron which were primarily defined on the basis that they showed adaptation in spike frequency and stopped firing prior to the cessation of the 500 ms suprathreshold current injection (+120pA; superimposed on −60pA step) similar to rAD interneurons (Butt et al., 2005). (H) reconstructed morphology of the cells whose electrophysiological profile is shown in panel (G). Complete profile of interneurons recorded from E10.5 (I) and E12.5 (J) control (blue histogram bars; E10.5 n=55, E12.5 n=25) and conditional loss of function (green; E10.5 n=55, E12.5 n=31) juvenile animals (P13–P21). FS, fast spiking interneuron; IB, intrinsic bursting interneuron; NFS, non-fast spiking interneuron; dNFS, delayed non-fast spiking interneuron; AD, adapting interneurons including iAD, sAD and rAD; bNA, burst nonadapting; LS, late spiking; IS, irregular spiking interneuron.