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. 2016 Sep 21;91(6):1260-1275.
doi: 10.1016/j.neuron.2016.08.020.

Transcriptional Networks Controlled by NKX2-1 in the Development of Forebrain GABAergic Neurons

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Free PMC article

Transcriptional Networks Controlled by NKX2-1 in the Development of Forebrain GABAergic Neurons

Magnus Sandberg et al. Neuron. .
Free PMC article

Abstract

The embryonic basal ganglia generates multiple projection neurons and interneuron subtypes from distinct progenitor domains. Combinatorial interactions of transcription factors and chromatin are thought to regulate gene expression. In the medial ganglionic eminence, the NKX2-1 transcription factor controls regional identity and, with LHX6, is necessary to specify pallidal projection neurons and forebrain interneurons. Here, we dissected the molecular functions of NKX2-1 by defining its chromosomal binding, regulation of gene expression, and epigenetic state. NKX2-1 binding at distal regulatory elements led to a repressed epigenetic state and transcriptional repression in the ventricular zone. Conversely, NKX2-1 is required to establish a permissive chromatin state and transcriptional activation in the sub-ventricular and mantle zones. Moreover, combinatorial binding of NKX2-1 and LHX6 promotes transcriptionally permissive chromatin and activates genes expressed in cortical migrating interneurons. Our integrated approach provides a foundation for elucidating transcriptional networks guiding the development of the MGE and its descendants.

Figures

Figure 1
Figure 1. Conditional Deletion of Nkx2-1 in the Embryonic MGE
(A–L) AI14 immunofluorescence (A–C, G–I) and Nkx2-1exon-2 in situ (D–F, J–L) on WT and Nkx2-1cKO at E9.5, E11.5, and E13.5. (M) Heatmaps showing relative expression and log2 fold change of dysregulated genes in the Nkx2-1cKO MGE at E13.5. (N and O) Functional enrichment analysis on down- (N) and up-regulated (O) genes in the Nkx2-1cKO MGE at E13.5. (P–S) In situ analysis using probes detecting the downregulated genes Tcf12 (P and Q) and Tgf3 (R and S) in WT and Nkx2-1cKO forebrain at E13.5. (T–W) In situ analysis using probes detecting the upregulated genes Fzd8 (T and U) and Id4 (V and W) in WT and Nkx2-1cKO forebrain at E13.5.
Figure 2
Figure 2. Genomic Characterization of NKX2-1 Binding in the MGE
(A) Biological replicates of NKX2-1 ChIP-seq showing consistent NKX2-1 binding at the 5′ end of Nrxn3. (B) Average profile of NKX2-1 binding at called ChIP-seq peaks comparing the three biological replicates. (C) NKX2-1 consensus motif derived from de novo motif analysis of NKX2-1 ChIP-seq data. (D) Genomic features of NKX2-1-bound REs. (E) Proportion of differentially expressed genes in the Nkx2-1cKO with TSS and distal NKX2-1 binding. Fisher’s exact test was used to test significance between the groups: ***p < 0.001, **p < 0.01. (F) Enriched gene ontology annotations of putative NKX2-1 binding target genes for distal REs (blue) and NKX2-1-bound TSSs (green).
Figure 3
Figure 3. Histone Profile of NKX2-1-Bound REs in the MGE
(A) Heatmaps showing H3K4me1, H3K4me3, H3K27ac, and H3K27me3 enrichment around NKX2-1-bound REs. REs divided into eight groups defined by their combined histone profile: (1) active TSS (1.1.1.0 = H3K4me1+, H3K4me3+, H3K27ac+, and H3K27me3−); (2) bivalent TSS (1.1.1.1); (3) active (strong) distal (1.1.1.0); (4) active (weak) distal (1.0.1.0); (5) bivalent distal (1.1.1.1); (6) latent distal (1.0.0.0); (7) repressed distal (1.0.0.1); and (8) no histone (0.0.0.0). (B) Average profile of H3K4me1, H3K4me3, H3K27me3, and H3K27ac at (3) active (strong) distal, (4) active (weak) distal, (5) bivalent distal, and (7) repressed distal REs. (C) Relative gene expression of closest TSS in relation to RE groups as defined in (A). Two-sample t tests were used to test significance compared to the control gene set: ***p < 0.001, **p < 0.01, and *p < 0.05.
Figure 4
Figure 4. Definition of aREs and rREs Based on Changes in the Histone Profile in the Nkx2-1cKO MGE
(A and B) Genomic regions of the Lhx8 (A) and Gli2 (B) loci with the ChIP-seq datasets and genomic features shown; NKX2-1 ChIP-seq, H3K4me3, H3K4me1, H3K27ac, H3K27me3, UCSC genes, and mammalian conservation. The H3 modification ChIP-seqs were performed on both WT and Nkx2-1cKO MGE at E13.5. Called TF binding peaks are labeled with an asterisk. (C–F) In situ analysis of Lhx8 (C and D) and Gli2 (E and F) in WT and Nkx2-1cKO forebrain at E13.5.
Figure 5
Figure 5. Identification of NKX2-1-Bound REs Mediating Transcriptional Activation and Repression
(A and B) Average profile of H3K27ac and H3K27me3 at aREs (A) and rREs (B) in WT and Nkx2-1cKO MGE at E13.5. (C) Proportion of TSS and distal NKX2-1-bound REs with changes in the chromatin profile in the Nkx2-1cKO in the E13.5 MGE. (D) Average fold change in expression of genes associated to distal aREs and rREs in the Nkx2-1cKO MGE at E13.5. Two-sample t test was used to test significance between the groups: ***p < 0.001. (E) Proportion of distal aREs and rREs with an associated increase or decrease in expression of the gene with closest TSS. (F–H) Proportion of background, TSS, all NKX2-1-bound Res, distal Res, distal aREs, and distal rREs with identified de novo motifs. All motifs (F) and manually curated list (G). Sequence of motifs and potential TF recognizing the motifs (H). Fisher’s exact test was used to test significance between the groups for (C) and (E). Wilcox test was used to compare expression distribution in (D): ***p < 0.001, **p < 0.01.
Figure 6
Figure 6. NKX2-1 and LHX6 Co-occupy aREs in the MGE
(A) Enrichment of primary LHX6 de novo motif in relation to the center of LHX6-bound REs. (B) Proportion of aREs in NKX2-1+ (all), NKX2-1+/LHX6+, and Nkx2-1+/Lhx6− bound REs. Chi-square test was used to test significance between the groups: ***p < 0.001. (C) Proportion of rREs in NKX2-1+ (all), NKX2-1+/LHX6+, and Nkx2-1+/Lhx6− bound REs. Fisher’s exact test was used to test significance between the groups: ***p < 0.001. (D and E) Genomic region of the Lhx6 (D) and Gas1 (E) loci (+/−100 kb from TSS); NKX2-1 ChIP-seq, LHX6 ChIP-seq, H3K4me1, H3K4me3, H3K27Ac, H3K27me3, UCSC genes, and mammalian conservation. Both WT and Nkx2-1cKO data are shown for the H3 modifications. Called peaks are labeled with an asterisk. (F–I) In situ analysis of Lhx6 (F and G) and Gas1 (H and I) transcription in WT and Nkx2-1cKO forebrain at E13.5.
Figure 7
Figure 7. LHX6 Is Required for Activating Transcription in the SVZ and MZ of the MGE
(A) Proportion of MGE, LGE, and cortex activity of +/−REs and +/+REs at E11.5. (B) VZ, SVZ, and MZ activity of +/+REs in the MGE at E11.5. Chi-square test was used to test significance between the groups: *p < 0.05. (C) Browser view of +/−REs with tracks; VISTA transgenic ID, NKX2-1 ChIP-seq, LHX6 ChIP-seq, H3K4me1, H3K4me3, H3K27ac, H3K27me3, UCSC genes, and mammalian conservation. Both WT and Nkx2-1cKO data are shown for H3 modifications. Called peaks are labeled with an asterisk. VISTA transgenics showing in vivo activity of +/−REs (hs187, hs271, and hs1336) at E11.5. (D) Browser view of +/+REs with tracks; VISTA transgenic ID, NKX2-1 ChIP-seq, LHX6 ChIP-seq, H3K4me1, H3K4me3, H3K27ac, H3K27me3, UCSC genes, and mammalian conservation. Both WT and Nkx2-1cKO data are shown for H3 modifications. Called peaks are labeled with an asterisk. VISTA transgenics showing in vivo activity of +/+REs (hs550, hs623, and hs883) at E11.5.
Figure 8
Figure 8. In Vivo Activity of NKX2-1 in the Developing MGE
(A) mm1429 locus with the ChIP-seq datasets and genomic features; NKX2-1 ChIP-seq, LHX6 ChIP-seq, H3K4me1, H3K4me3, H3K27ac, H3K27me3, UCSC genes, and mammalian conservation. Both WT and Nkx2-1cKO data are shown for H3 modifications. Called peaks are labeled with an asterisk. (B) mm1429 transgenic showing activity in the SVZ and MZ of the MGE at E12.5. (C) Schematic of mm1429 with NKX2-1 and LHX6 motifs. Luciferase reporter assay showing the opposing effects of mutating NKX2-1 and LHX6 motifs in mm1429. Data are represented as mean ± SEM (n = 3). (D–I) Genomic regions of the Gbx1 (D) and the Gbx2 (E) loci with the same ChIP-seq datasets and genomic features shown in Figure 5A. In situ analysis of Gbx1 (F and G) and Gbx2 (H and I) transcription in WT and Lhx6; Lhx8dKO forebrain at E13.5. (J) Summary model showing the combined activity of NKX2-1 and LHX6 at REs in the VZ and SVZ.

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