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First published online 13 March 2008
doi: 10.1242/dev.015123

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NKX2.1 specifies cortical interneuron fate by activating Lhx6

Department of Psychiatry, and Department of Neurology and Neuroscience, Weill Medical College of Cornell University, 1300 York Avenue, Box 244, New York, NY 10021, USA.

View larger version (75K): [in this window] [in a new window]   Fig. 1. Transfection of Nkx2.1-/- slices with Nkx2.1 cDNA results in rescue of interneuron phenotypes. (A-D) Schematic showing slice electroporation and transplantation paradigm. (A) The Nkx2.1 domain is shown in the MGE of an E12.5 wild-type (wt) mouse embryo slice. The MGE*, a region that expresses a truncated Nkx2.1 transcript (Sussel et al., 1999), is shown in the slice from an Nkx2.1 mutant embryo. (B) This MGE* region is targeted for electroporation, and after 1 day in vitro (DIV) the region is dissected out, dissociated and transplanted (C,D) directly into the neocortex of neonatal pups (as in H-N), or plated onto a high-density culture of neonatal cortical cells [as in Xu et al. (Xu et al., 2004); see Fig. S2 in the supplementary material]. (E,F) Coronal sections of a slice from an E12.5 wt embryo that was electroporated with pNkx2.1-GFP, maintained 1DIV, then fixed and examined for GFP fluorescence (E) and NKX2.1 immunolabeling (F). The right-hand, electroporated side of the slice has extensive ectopic NKX2.1 expression, whereas only native NKX2.1 expression is seen on the left-hand side of the slice (arrow in F). (G) A slice from an Nkx2.1-/- embryo was electroporated with pNkx2.1-GFP. After 1DIV, cells from the MGE* (outlined in white) were transplanted into the cortical plate of a neonatal pup and then examined at postnatal day 30 (P30) in 40 µm coronal sections. (H) Transplanted GFP-expressing cells scattered through the medial cortex. (I-N) Examples of co-labeling for GFP and parvalbumin (PV; I,J), somatostatin (SST; K,L), and neuropeptide Y (NPY; M,N). In control experiments with pGFP vector, almost no cells expressing any of these markers are detected after transplantation of Nkx2.1-/- MGE* progenitors (Table 1). MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; Ctx, cerebral cortex. Scale bars: 100 µm in G,H.

View larger version (41K): [in this window] [in a new window]   Fig. 2. RNAi knockdown of Lhx6 blocks the rescue of PV- or SST-expressing cells by Nkx2.1. (A) The results of RT-PCR for Nkx2.1 (lanes 1 and 2) and Lhx6 (lanes 3 and 4) from the MGE-like region (MGE*) of an E12.5 Nkx2.1-/- slice that was electroporated with pGFP control vector (lanes 1 and 3) or pNkx2.1-GFP (lanes 2 and 4). Lhx6 (422 bp band in lane 4) is induced in the Nkx2.1-transfected slice. (B,C) Transfected cells from the MGE* of Nkx2.1-/- slices that were cultured from E12.5+1DIV, then transplanted into the parietal cortex of a neonatal mouse in vivo and evaluated in 40 µm sections at P30. The neuron in B was from a transplant that received pNkx2.1+ scramble RNAi control, and co-immunolabels for LHX6 (red) and somatostatin (SST, blue pseudocolored from Cy5 signal). Transfection of pNkx2.1-GFP + shLhx6 RNAi blocks the induction of Lhx6 and blocks rescue of the SST+ phenotype (C). Note that endogenous SST+ GFP-negative cells in the mouse cortex co-label for LHX6. (D) Quantification of the effect of shLhx6 RNAi on the Nkx2.1 rescue of PV+ and SST+ interneuron fate (n=3 donor samples for each condition transplanted into separate pups, Student's t-test, **P<0.01, *P<0.03).

View larger version (84K): [in this window] [in a new window]   Fig. 3. Lhx6 expression can rescue interneuron phenotypes in transplanted cells from Nkx2.1-/- MGE*. pLhx6-GFP was electroporated into the MGE-like region of E12.5 Nkx2.1-/- slices, then after 1DIV the transfected regions were dissociated and transplanted into the cortex of neonatal pups. Shown are coronal sections through a P30 mouse that had received the transplantation into the cortical plate at P1. (A-H) Examples of co-labeling for GFP together with Kv3.1 and parvalbumin (PV; A-D), somatostatin (SST; E,F), and neuropeptide Y (NPY; G,H). In control experiments with pGFP vector, few cells expressing these markers are detected after transplantation of Nkx2.1-/- MGE-like progenitors (see text and Table 1). (I,J) Transfected neurons (I, pGFP control; j, pLhx6-GFP) photographed at higher magnification to reveal dendritic spines. Insets show the boxed regions at higher magnification. (K) The frequency of heavily spiny neurons is significantly lower in the Nkx2.1-/- MGE* cells transfected with Lhx6 than in controls (41.9% versus 24.7%, n=3, *P<0.03). In addition, those Nkx2.1-/- cells `rescued' for expression of PV or SST by Lhx6 are nearly all non- or sparsely spiny. These results suggest that Lhx6 can act downstream of Nkx2.1 to direct some aspects of both the neurochemical and morphological fates of MGE-derived cortical interneurons.

View larger version (44K): [in this window] [in a new window]   Fig. 4. Binding of NKX2.1 to a conserved consensus binding sequence in the Lhx6 promoter. (A) Evolutionarily conserved region (ECR) visualization of the LHX6 gene locus in the human genome. The conservation profile of the human sequence in comparison with the mouse, chicken, frog and fugu genomes is shown. In the model, the Lhx6 loci of the given species are displayed on the horizontal axis and the vertical axis represents the percentage of base-pair identity (from 50 to 100%) between the given species and human. The horizontal bar above each species provides an overview of the distribution of ECRs. A conserved alignment is blue if it overlaps with a coding exon. Yellow, untranslated region; pink, intron (although most of the pink region 5' to the Lhx6 translation start site in fact appears to be an intergenic region, see NCBI sequence data); red, intergenic region. The green bars at the bottom indicate repetitive elements in the sequence. (B) rVISTA analysis revealed a conserved NKX2.1/TITF1 binding site 240 bp from the translation start site. The arrowheads indicate the locations of the PCR primers used to clone a 2.1 kb promoter fragment of genomic DNA (see Materials and methods and Figs 5, 6). (C) The NKX2.1 consensus binding sequence is located within a 119 bp PCR product that was used to probe chromatin immunoprecipitation results on E12.5 MGE. IgG, control mouse IgG; Nkx2.1 Ab, mouse anti-NKX2.1; Input, control PCR on a crosslinked, sonicated MGE sample; left, molecular weight marker. These results suggest that NKX2.1 binds the Lhx6 promoter at a highly conserved NKX2.1 consensus binding sequence in vivo.

View larger version (112K): [in this window] [in a new window]   Fig. 5. Nkx2.1 activates the expression of an Lhx6 promoter reporter. Shown are examples of coronal, telencephalic slices at E13.5+1DIV that were electroporated with the constructs indicated. (A-C) Constitutively expressing pCAG-DsRed2 (A, pDsRed2) was introduced into a wt mouse embryo slice together with a reporter construct that contains 2.1 kb of the Lhx6 promoter region placed 5' to IRES-GFP (p5'-Lhx6-GFP, B). The merged image in C shows that the reporter construct is detectable in the ventral, Nkx2.1-expressing region (arrowheads) and not in the electroporated region of the medial cortex (arrow). (D-F) In marked contrast to B and C, electroporation of p5'-Lhx6-GFP into the MGE-like region (MGE*) of this slice from an Nkx2.1 null results in no reporter expression (E,F). (G-I) However, the expression of p5'-Lhx6-GFP is rescued in an Nkx2.1-null slice by the addition of exogenous Nkx2.1 (red signal in G and I is NKX2.1 immunofluorescence). (J-L) A wild-type slice electroporated with a mutated reporter construct in which only the NKX2.1 consensus binding sequence has been deleted (p5'--Lhx6-GFP; see Materials and methods). Minimal expression of GFP is detected in the MGE with this construct (K-L). n=at least five experiments for each result. MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; Ctx, cerebral cortex. Scale bar: 200 µm in A for A-L.

View larger version (99K): [in this window] [in a new window]   Fig. 6. Ectopic activation of Lhx6-GFP reporter expression by Nkx2.1. (A-C) In mouse embryo slice cultures maintained from E13.5+1DIV, no expression of GFP is detected when p5'-Lhx6-GFP is introduced into the wt LGE (arrowhead) or lateral cortex (arrow). (D-F) Ectopically expressed NKX2.1 drives Lhx6-reporter expression in these regions. (G-I) This activation is not present in response to ectopic expression of an altered NKX2.1 construct containing a missense point mutation in the homeodomain that abrogates its ability to bind DNA (Krude et al., 2002). Note that the red signal in G and I is NKX2.1 immunofluorescence that is not affected by the point mutation. (J-L) Co-electroporation of pNkx2.1 and the Lhx6 promoter reporter construct that lacks the NKX2.1 consensus binding sequence (p5'-Lhx6-GFP) results in little expression of GFP. (M-O) As with Nkx2.1 cDNA, fusion of the VP16 activator domain to Nkx2.1 strongly induces the reporter GFP expression in wt LGE or cerebral cortex. In this case, there is reduced detection of the altered NKX2.1 protein by immunofluorescence (M,O). MGE, medial ganglionic eminence; LGE, lateral ganglionic eminence; Ctx, cerebral cortex. Scale bar: 200 µm in A for A-O.

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