Necdin promotes GABAergic neuron differentiation in cooperation with Dlx homeodomain proteins

Abstract

Necdin, a member of the MAGE (melanoma antigen) protein family, is expressed predominantly in terminally differentiated neurons. The necdin gene NDN is maternally imprinted and expressed only from the paternal allele, the deficiency of which is implicated in the pathogenesis of the neurodevelopmental disorder Prader-Willi syndrome. Necdin binds to its homologous MAGE protein MAGE-D1 (also known as NRAGE or Dlxin-1), which interacts with Msx (msh homeobox) and Dlx (distal-less homeobox) family homeodomain transcription factors. Members of the Dlx homeobox gene family are involved in the differentiation and specification of forebrain GABAergic neurons. Here we demonstrate that necdin associates with Dlx homeodomain proteins via MAGE-D1 to promote the differentiation of GABAergic neurons in mouse embryonic forebrain. Immunohistochemical analysis revealed that necdin was coexpressed with Dlx2, Dlx5, or MAGE-D1 in a subpopulation of embryonic forebrain cells. Necdin bound to Dlx2 and Dlx5 via MAGE-D1 and enhanced Dlx2-dependent activation of the Wnt1 (wingless-type MMTV integration site family) promoter. Necdin significantly increased the populations of cells expressing the GABAergic neuron markers calbindin D-28k and glutamic acid decarboxylase when overexpressed by electroporation in cultured forebrain slices. In this assay, Dlx5N, a truncated Dlx5 mutant that competes with Dlx2 to bind MAGE-D1, diminished the effect of necdin on GABAergic neuron differentiation. Furthermore, mutant mice lacking the paternal necdin allele showed a significant reduction in the differentiation of forebrain GABAergic neurons in vivo and in vitro. These results suggest that paternally expressed necdin facilitates the differentiation and specification of GABAergic neurons in cooperation with Dlx homeodomain proteins.

Figure 1.

Necdin is coexpressed with Dlx2, Dlx5, and MAGE-D1 in mouse embryonic forebrain. A, Double immunostaining for necdin and Dlx2, Dlx5, or MAGE-D1 (D1). E13.5 forebrain sections were double immunostained for necdin (green) and Dlx2 (red), Dlx5 (red), or MAGE-D1 (red), and two images were merged (yellow). CX, Cortex; LV, lateral ventricle; LGE, lateral ganglionic eminence; SP, septum. B, C, Enlarged images (boxed in A) of the septum (B) and cortex (C). Images of double immunostaining for necdin and calbindin D-28k (Calbindin) or GAD (B) and for necdin and calbindin or calretinin (C) are also shown. D, Double immunostaining for necdin and Dlx2 or MAGE-D1 in the hypothalamus. Enlargements of the hypothalamus (boxed region in top panels) are shown in bottom panels. Scale bars: A, 200 μm; B, C, top two rows in D, 10 μm; bottom two rows in D, 100 μm. Arrowheads in B–D indicate representative double-labeled cells.

Figure 2.

Interactions of MAGE-D1 with Dlx proteins in vitro and in vivo. A–C, In vitro binding assay. MAGE-D1 (D1) deletion mutants fused to MBP were separated by 10% SDS-PAGE and visualized by Coomassie brilliant blue staining (B). MBP fusion proteins immobilized on amylose resin were incubated with GST-Dlx2 (GDlx2) or GST-Dlx5 (GDlx5), and bound proteins were detected with anti-GST antibody (C). Molecular sizes are in kilodaltons. The results are schematized in A. IRD, Interspersed hexapeptide repeat domain; MHD, MAGE homology domain; NBD, necdin binding domain. D, Immunoprecipitation assay. Lysates (400 μg) of HEK293 cells transfected with FLAG-Dlx2 and FLAG-Dlx5 cDNAs were immunoprecipitated (IP) and immunoblotted (IB) with anti-MAGE-D1 antibody (αD1) and anti-FLAG antibody M2 (αFLAG). Expressed proteins (10 μg of lysate) are in the bottom two panels. E, Immunoaffinity assay. Tissue lysate (1 mg) of E13.5 forebrain was applied to immunoaffinity columns carrying αD1 IgG and preimmune IgG (Pre IgG). Bound proteins were analyzed by Western blotting for Dlx2, Dlx5, necdin, PCNA, and MAGE-D1. Lysate, Tissue lysate (30 μg). F, In vitro binding assay. MBP-D1 and MBP-Dlx5N fusions (bottom) immobilized on amylose resin were incubated with GST-Dlx2, and bound proteins were detected with anti-GST antibody (top). G, Immunoprecipitation assay for competition between Dlx2 and Dlx5N. Lysates (400 μg) of HEK293 cells transfected with FLAG-Dlx2 and FLAG-Dlx5N (amino acids 1–132) cDNAs were immunoprecipitated with αD1 and immunoblotted with αFLAG. Expressed proteins (10 μg of lysate) are in the bottom three panels.

Figure 3.

Formation of a ternary complex containing necdin, MAGE-D1, and Dlx2. A, Coimmunoprecipitation assay. Lysates (400 μg) of HEK293 cells transfected with cDNAs for FLAG-Dlx2, FLAG-Dlx5, and necdin were immunoprecipitated (IP) and immunoblotted (IB) with anti-MAGE-D1 (αD1), αFLAG, and α-necdin antibodies. Expressed proteins (10 μg of lysate) are in the bottom three panels. B, Immunoaffinity assay. E13.5 forebrain lysate (1 mg) was applied to immunoaffinity columns carrying α-necdin IgG and preimmune IgG (Pre IgG), and bound proteins were analyzed by Western blotting for MAGE-D1, Dlx2, Dlx5, PCNA, and necdin. Lysate, Tissue lysate (30 μg). C, In vitro binding assay. Purified proteins were immunoprecipitated with α-necdin (C2) and immunoblotted with αDlx2 and αD1. Proteins in 5% input are in the bottom three panels. D, Immunoprecipitation assay for competition between Dlx2 and Dlx5N. Lysates (400 μg) of HEK293 cells transfected with necdin, FLAG-Dlx2, and FLAG-Dlx5N cDNAs were immunoprecipitated with α-necdin (NC243) and immunoblotted with αFLAG (bands at 48 kDa for FLAG-Dlx2 and 23 kDa for FLAG-Dlx5N) and αD1 (85 kDa for endogenous D1). Expressed proteins (10 μg of lysate) are in the bottom four panels. E, Promoter assay. Combinations of expression vectors for FLAG-Dlx2 (Dlx2, 0.25 μg), necdin (0.6 μg), Myc-tagged MAGE-D1 (D1, 0.6 μg), and FLAG-Dlx5N (Dlx5N, 0.6 μg) were transfected into HEK293 cells with the promoter-reporter vector pGL2-WIP (1 μg) carrying Wnt1 promoter (WIP). The total amount of plasmids was adjusted to 5 μg/assay by adding the empty vector. The luciferase activity was measured with a luminometer (mean ± SEM; n = 3).

Figure 4.

Promotion of GABAergic neuron differentiation by necdin. A, B, Immunohistochemistry. Coronal sections of E13.5 mouse forebrain were double immunostained for necdin and Dlx2 (A). Forebrain slices were cultured for 24 h, electroporated with GFP cDNA, and incubated for an additional 48 h. Transfected cells are visualized by GFP immunostaining (GFP) (B). CX, Cortex; LV, lateral ventricle; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence. Scale bars, 200 μm. C, D, Electroporation assay in the ganglionic eminences. E13.5 forebrain slices were transfected by electroporation with expression vectors for GFP, necdin, and MAGE-D1 (D1). GABAergic neuron differentiation in lateral ganglionic eminence and medial ganglionic eminence (area encircled with the yellow line in B) was analyzed by double immunostaining for GFP (green) and GAD (red) or calbindin (red) (C). Shown are merged images (yellow). Arrowheads indicate representative GFP-positive cells that express GAD or calbindin (yellow). Scale bar, 40 μm. GFP-positive cells expressing calbindin or GAD were observed with a confocal laser microscope and counted (examined >500 GFP-positive cells per slice; mean ± SEM; n = 4) (D). *p < 0.01. E, Western blot analysis. Calbindin, GAD, MAGE-D1, necdin, Dlx2, and tubulin in transfected forebrain region (encircled with the red line in B) were analyzed by Western blotting. F, Electroporation assay in the cortex. Expression vectors for GFP, necdin, and Dlx2 were transfected into the cortex (encircled with the blue line in B), and cells expressing both GFP and GAD were counted after double immunostaining (examined >300 GFP-positive cells per slice; mean ± SEM; n = 3). *p < 0.01.

Figure 5.

Suppression of necdin-induced GABAergic neuron differentiation by Dlx5N. A, B, Electroporation assay in the ganglionic eminences. E13.5 mouse forebrain slices were electroporated with expression vectors for GFP, necdin, and Dlx5N as in Figure 4. Cells expressing calbindin and GAD were detected by immunohistochemistry (A) and counted (B) (mean ± SEM; n = 3; *p < 0.01). C, D, Electroporation assay in the septum. E13.5 mouse forebrain slices were transfected with expression vectors for GFP and Dlx5N. Transfected cells expressing GAD in the septum were detected by immunohistochemistry (C) and quantified as in Figure 4 (mean ± SEM; n = 4). **p < 0.003. Scale bars, 40 μm.

Figure 6.

Reduction in GABAergic neuron differentiation in the forebrain of necdin-deficient mouse embryos. A, B, Western blot analysis. Proteins in forebrain lysates from wild-type (Ndn^+m/+p) and necdin-deficient (Ndn^+m/−p) embryos at E14.5 were analyzed by Western blotting. Each lane represents the extract from one littermate (A). The signal intensities relative to those of β-tubulin are presented (mean ± SEM; n = 5 per each group) (B). *p < 0.01, **p < 0.001. C–E, Immunohistochemical assay. Forebrain sections were immunostained for Dlx2 and Dlx5 (C). Enlarged images are shown in the insets (boxed regions). Immunoreactivities of calbindin, GAD, and GABA in the septum are also shown in D. Immunopositive cells were counted and presented as the number per square millimeter (E) (mean ± SEM; n = 4 for calbindin; n = 3 for GAD, GABA, Dlx5, and Dlx2; examined 5 sections per embryo). Scale bars: C, 100 μm; insets in C, D, 20 μm. *p < 0.05; **p < 0.02; ***p < 0.001. NS, Not significant (p > 0.05).

Figure 7.

Reduction in GABAergic neuron differentiation in primary cultures prepared from necdin-deficient mouse embryos. A, B, Immunocytochemical assay. Cells were prepared from the ganglionic eminences of wild-type (Ndn^+m/+p) and necdin-deficient (Ndn^+m/−p) embryos at E14.5 and cultured for 4 d. Primary cells were double immunostained for MAP2 and calbindin, GAD, Dlx5, or Dlx2 (A). Arrowheads point to representative double-immunopositive cells. Scale bar, 20 μm. MAP2-positive cells (neurons) expressing calbindin, GAD, Dlx5, and Dlx2 were counted (examined >200 neurons per culture for GAD, Dlx5, and Dlx2, >1500 for calbindin; mean ± SEM; n = 3) (B). *p < 0.05; **p < 0.01; ***p < 0.001. NS, Not significant (p > 0.05).