Postmitotic Nkx2-1 controls the migration of telencephalic interneurons by direct repression of guidance receptors

Abstract

The homeodomain transcription factor Nkx2-1 plays key roles in the developing telencephalon, where it regulates the identity of progenitor cells in the medial ganglionic eminence (MGE) and mediates the specification of several classes of GABAergic and cholinergic neurons. Here, we have investigated the postmitotic function of Nkx2-1 in the migration of interneurons originating in the MGE. Experimental manipulations and mouse genetics show that downregulation of Nkx2-1 expression in postmitotic cells is necessary for the migration of interneurons to the cortex, whereas maintenance of Nkx2-1 expression is required for interneuron migration to the striatum. Nkx2-1 exerts this role in the migration of MGE-derived interneurons by directly regulating the expression of a guidance receptor, Neuropilin-2, which enables interneurons to invade the developing striatum. Our results demonstrate a role for the cell-fate determinant Nkx2-1 in regulating neuronal migration by direct transcriptional regulation of guidance receptors in postmitotic cells.

Figure 1. Cortical interneurons downregulate Nkx2-1 protein expression after leaving the MGE progenitor zone

(A) Coronal section through the brain of an E13.5 mouse embryo showing Nkx2-1 protein expression in the medial ganglionic eminence (MGE), striatum (Str), globus pallidus (GP), preoptic area (POA) and septum (Se). (B and C) Higher magnification images of the areas boxed in (A). (D–E’) MGE ventricular zone (VZ) from an E13.5 embryo depicting MGE cells stained with Nkx2-1 and propidium iodine (PI) (D–D’), and MGE progenitors stained S-phase marker BrdU and Nkx2-1 (E–E’). (F–H) Expression of Nkx2-1 protein in GFP-expressing cells after transplantation of GFP-expressing progenitors in the MGE. As illustrated in the schematic diagram (F’) and the high magnification images (G and H), the majority of migrating cells had undetectable levels of Nkx2-1 (green dots), many cells expressed low levels of Nkx2-1 (yellow dots with green circle), and a small number of cells expressed high levels of Nkx2-1 protein (yellow dots). The numbers depicted in the schematic (F’) describe the location of cells shown in (G) and (H). H, hippocampus; LGE, lateral ganglionic eminence; LV, lateral ventricle; NCx, neocortex. Asterisk, transplant. Scale bars equal 200 µm (A and F), 50 µm (B, C, G and H), and 10 µm (D–E’).

Figure 2. Nkx2-1 overexpression in MGE-derived interneurons prevents their migration to the cortex

(A and B) Schematic diagrams of the focal electroporation experiment and the Nkx2-1 (372 amino acids) constructs used in these experiments. (C–G) Migration of MGE-derived cells electroporated with Gfp (C) or with Gfp and Nkx2-1 (D), Nkx2-1^ΔTN (E), Nkx2-1^ΔCt (F) or Nkx2-1^A35T (G). Arrowheads point to cells that have reached the cortex. Dotted lines indicate the limits of the organotypic slices. (H) Schematic representation of the migratory routes adopted by MGE-derived cells electroporated with Gfp and Gfp + Nkx2-1^A35T (green arrow) or with Gfp Nkx2-1, Nkx2-1^ΔTN or Nkx2-1^ΔCt (black dotted arrow). GP, globus pallidus; H, hippocampus; HD, homeodomain; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; NCx, neocortex; NK2-SD, NK2 specific domain; PCx, piriform cortex; POA, preoptic area; Str, striatum; TN, Tinman motif. Scale bar equals 200 µm.

Figure 3. Reduced numbers of striatal interneurons after postmitotic loss of Nkx2-1function

(A, B, D, E, G, H, J and K) Coronal sections through the telencephalon of E15.5 control (A, D, G and J) and Lhx6-Cre;Nkx2-1^Fl/Fl mutant (B , E, H and K) embryos showing Lhx6 (A and B), Lhx7 (D and E), Er81 (G and H) and Sst (J and K) mRNA expression. (C, F, I and L) Quantification of the number of Lhx6, Lhx7, Er81 and Sst-expressing cells in the striatum of E15.5 control and Lhx6-Cre;Nkx2-1^Fl/Fl mutant embryos. Histograms show average ± s.e.m. 1083.96 ± 23.47 (Lhx6 control); 862.07 ± 49.01 (Lhx6 mutant); 452.32 ± 21.78 (Lhx7 control); 212.02 ± 18.68 (Lhx7 mutant); 601.60 ± 12.74 (Er81 control); 397.05 ± 23.84 (Er81 mutant); 579.03 ± 39.17 (Sst control); 432.18 ± 20.03 (Sst mutant). *** p < 0.001, ** p < 0.01 and * p < 0.05, t-test. ec, external capsule; Str, striatum. Scale bar equals 100 µm.

Figure 4. Loss of Nkx2-1 function decreases the number of interneurons in the postnatal striatum

(A, B, D, E, G and H) Coronal sections through the striatum of P25 control (A, D and G) and Lhx6-Cre;Nkx2-1^Fl/Fl mutant (B , E and H) mice showing ChAT (A and B), PV (D and E) and SST (G and H) expression. (C, F and I) Quantification of the number of ChAT, PV and SST-expressing cells in the striatum of P25 control and Lhx6-Cre;Nkx2-1^Fl/Fl mutant mice. Histograms show average ± s.e.m. 39.42 ± 2.26 (ChAT control); 10.94 ± 1.04 (ChAT mutant); 46.32 ± 4.52 (PV control); 14.71 ± 1.77 (PV mutant); 48.23 ± 1.65 (SST control); 41.73 ± 2.81 (SST mutant). *** p < 0.001 and ** p < 0.01, t-test. CPu, caudate putamen. Scale bar equals 100 µm.

Figure 5. Tracing experiments reveal less interneurons invading the striatum after postmitotic loss of Nkx2-1 function

(A and B) Coronal sections through the striatum of P0 Lhx6-Cre;Nkx2-1^Fl/+;Rosa-YFP control (A) and Lhx6-Cre;Nkx2-1^Fl/Fl;Rosa-YFP mutant (B) mice showing YFP expression. YFP is also detected in scattered blood vessels, as previously reported (Fogarty et al., 2007). (C) Quantification of the number of YFP-expressing cells in the striatum of P0 Lhx6-Cre;Nkx2-1^Fl/+;Rosa-YFP control and Lhx6-Cre;Nkx2-1^Fl/Fl;Rosa-YFP mutant mice. Histograms show average ± s.e.m. 889.31 ± 79.03 (YFP control); 664.12 ± 58.40 (YFP mutant). * p < 0.05, t-test. (D) Schematic diagram of the focal electroporation experiment. The number of Gfp-expressing cells was counted in a fixed volume of the cortex and striatum (black boxes) and the ratio between these populations was determined for each slice (E–F) Migration of MGE-derived cells in E13.5 Lhx6-Cre;Nkx2-1^Fl/+;Rosa-YFP control (E) and Lhx6-Cre;Nkx2-1^Fl/Fl;Rosa-YFP mutant (F) slices. Occasionally, Gfp-expressing cells accumulated in the piriform cortex of mutant slices. Dotted lines indicate the limits of the organotypic slices. (G) Ratio of Gfp-expressing cells in the quantified region of the cortex and striatum for each individual E13.5 Lhx6-Cre;Nkx2-1^Fl/+;Rosa-YFP control and Lhx6-Cre;Nkx2-1^Fl/Fl;Rosa-YFP mutant slices. Average ± s.e.m. 2.55 ± 0.41 (control); 6.80 ± 1.22 (mutant). *** p < 0.001, t-test. Number of Gfp-expressing cells in the striatum (30.38 ± 4.03, control; 12.20 ± 3.80, mutant. *** p < 0.001, t-test) and cortex (76.80 ± 12.76, control; 84.20 ± 21.20, mutant) of electroporated slices. CPu, caudate putamen; H, hippocampus; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; NCx, neocortex; PCx, piriform cortex; Str, striatum. Scale bar equals 100 µm (A and B) and00 µm (E and F).

Figure 6. Postmitotic Nkx2-1 expression suppresses Sema3A/3F-mediated repulsion in MGE-derived interneurons

(A) Schematic diagram of the experimental paradigm used in MGE/COS co-culture confrontation assays. E13.5 slices were focally electroporated with Gfp, Nkx2-1-IRES-Gfp or Gfp + Nkx2-1^A35T. MGE explants were dissected from the electroporated region, confronted to DsRed or DsRed + Sema3A/3F-transfected COS cells aggregates and cultured in Matrigel® matrices. (B–G) Migration of Gfp (B and C) Nkx2-1-IRES-Gfp (D and E), and Gfp + Nkx2-1^A35T (F and G) electroporated MGE-derived cells in response to mock-transfected (B, D and F) or Sema3A/3F-transfected (C, E and G) COS cells aggregates. (H) Quantification of co-culture confrontation assays. P and D, proximal and distal quadrants, respectively. Histograms show average ± s.e.m. 1.13 ± 0.12 (Gfp MGE cells, mock-COS cells); 0.40 ± 0.05 (Gfp MGE cells, Sema3A/3F- COS cells); 1.11 ± 0.12 (Nkx2-1IRES-Gfp MGE cells, mock-COS cells); 0.97 ± 0.16 (Nkx2-1IRES-Gfp MGE cells, Sema3A/3F -COS cells); 1.10 ± 0.12 (Gfp + Nkx2-1A35T MGE cells, mock-COS cells); 0.52 ± 0.17 (Gfp + Nkx2-1A35T MGE cells, Sema3A/3F -COS cells). *** p < 0.001, t-test. MGE, medial ganglionic eminence; NCx, neocortex. Scale bar equals 50 µm.

Figure 7. Nkx2-1 represses Neuropilin-2 expression in MGE-derived cells

(A) Schematic diagram of the experimental paradigm used to isolate RNA from migrating MGE-derived cells. (B–C’) A Gfp-electroporated MGE explant stained with DAPI after 48 h in culture, before (B and B’) and after (C and C’) removing the explant core, which contains progenitor cells. (D) Semi-quantitative RT-PCR analysis comparing gene expression in Gfp- and Nkx2-1-electroporated MGE-derived cells. Negative (-, all reagents except cDNA) and positive (+, E14.5 MGE cDNA) controls were included in each run. Amplicon and molecular marker (M) base pairs (bp) are shown at the left and right sides of the panels, respectively. The Lhx6 gene has two transcripts: Lhx6 or Lhx6.1a (408 bp) and Lhx6.1b (306 bp). GAPDH was used as loading control. (E) Quantitative RT-PCR analysis for Neuropilin-1 and Neuropilin-2 expression in Gfp-and Nkx2-1-electroporated MGE-derived cells. Histograms show average ± s.e.m. 1.00 ± 0.25 (Gfp cells, Nrp1); 0.89 ± 0.11 (Nkx2-1 cells, Nrp1); 1.00 ± 0.19 (Gfp cells, Nrp2); 0.46 ± 0.29 (Nkx2-1 cells, Nrp2). * p < 0.05, t-test. MGE, medial ganglionic eminence; NCx, neocortex. Scale bar equals 50 µm.

Figure 8. Nkx2-1 binds the Neuropilin-2 promotor in vivo and regulates its expression

(A) Putative Nkx2-1 DNA binding sites [red and black boxes indicate 8/9 and 6-base pairs (bp) consensus sequences, respectively] in Nrp2-region2 (from -21375 bp 5’-CTTGC-3’ to -21086 bp 5’-GTGCT-3’) and Nrp2-region1 (from -327 bp 5’-CCGGA-3’ to -68 bp 5’-GGGGA-3’) of the Neuropilin-2 locus. (B) ChIP assays were performed using E13.5 MGE cells and a non-specific rabbit anti-IgG (Rb IgG) or a polyclonal antibody against Nkx2-1. Input chromatin represents 1% of the total chromatin. Negative (-, all reagents except DNA) and positive (+, E13.5 mouse genomic DNA) controls were included in each run. Nrp2-region 2 and Nrp2-region 1 amplicon size (bp, base pairs) are indicated. (C) The intensity of each PCR band was quantified and normalized against the input band. Histograms show average ± s.e.m. For Nrp2-region2: 0.29 ± 0.17 (Rb IgG) and 0.07 ± 0.06 (Nkx2-1). For Nrp2-region1: 0.06 ± 0.02 (Rb IgG) and 0.52 ± 0.06 (Nkx2-1). ** p < 0.01, t-test. (D) A luciferase reporter plasmid containing the Nrp2-region1 sequence upstream of the c-fos minimal promoter driving luciferase (pGL3-Nrp2-cfos-Luc) was co-transfected with either mock, Nkx2-1 or Nkx2-1^A35T expression vectors. For each condition, the relative luciferase activity corresponds to the ratio of normalized activities from the promoter-luciferase (pGL3-Nrp2-cfos-Luc) and empty-luciferase (pGL3-cfos-Luc) reporter vectors. Histograms show average ± s.e.m. 1.00 ± 0.09 (control), 0.64 ± 0.07 (Nkx2-1), and 0.89 ± 0.13 (Nkx2-1^A35T). ** p < 0.01, * p < 0.05, one-way ANOVA followed by Tukey’s post-test.