The polymicrogyria-associated GPR56 promoter preferentially drives gene expression in developing GABAergic neurons in common marmosets

Abstract

GPR56, a member of the adhesion G protein-coupled receptor family, is abundantly expressed in cells of the developing cerebral cortex, including neural progenitor cells and developing neurons. The human GPR56 gene has multiple presumptive promoters that drive the expression of the GPR56 protein in distinct patterns. Similar to coding mutations of the human GPR56 gene that may cause GPR56 dysfunction, a 15-bp homozygous deletion in the cis-regulatory element upstream of the noncoding exon 1 of GPR56 (e1m) leads to the cerebral cortex malformation and epilepsy. To clarify the expression profile of the e1m promoter-driven GPR56 in primate brain, we generated a transgenic marmoset line in which EGFP is expressed under the control of the human minimal e1m promoter. In contrast to the endogenous GPR56 protein, which is highly enriched in the ventricular zone of the cerebral cortex, EGFP is mostly expressed in developing neurons in the transgenic fetal brain. Furthermore, EGFP is predominantly expressed in GABAergic neurons, whereas the total GPR56 protein is evenly expressed in both GABAergic and glutamatergic neurons, suggesting the GABAergic neuron-preferential activity of the minimal e1m promoter. These results indicate a possible pathogenic role for GABAergic neuron in the cerebral cortex of patients with GPR56 mutations.

Figure 1

Generation of 0.3k hGPR56 e1m-EGFP transgenic marmosets. (A) Schematic diagram of the lentiviral vector. CMV; Cytomegalovirus promoter, ψ; packaging signal, RRE; rev responsive element, cPPT; central polypurine tract, WPRE: Woodchuck hepatitis virus Posttranscriptional Regulatory Element, hGPR56; 0.3-kbp human GPR56 e1m cis-element (minimal promoter). (B) The transgenic founder infants; I651TgF (female) and I757TgM (male). (C) Detection of genomic integration of transgene by genomic PCR. EGFP encoding sequence was amplified using template genomic DNA purified from hair cells of or placentas delivered with infants. Beta-actin amplifications were used as control. (D) Detection of transgene expression by RT-PCR. EGFP encoding sequence was amplified from cDNA templates prepared from RNAs purified from hair cells or placentas delivered with them with (RT+) or without (RT−) reverse transcriptase. Beta-actin amplifications were used as control.

Figure 2

Expression of GPR56 mRNA in the brain of wild type marmoset embryo. (A) Coronal sections of transgenic marmoset brain at the fourteenth embryonic week (EW) were hybridized with anti-sense (a–e) or sense probe (c) for GPR56 mRNA. Cerebral cortex (Cx), caudate nucleus (Cd), ventricular zone of the caudate (vCd), subventricular zone of the caudate (svCd), thalamus (Th), hippocampus (HIP), pons, midbrain (MB), ventricular zone (VZ), inter subventricular zone (iSVZ), outer subventricular zone (oSVZ), and intermediate zone (IZ) are indicated. Scale bars = 1 mm. (B) Enlarged image of the area marked by a square in panel b in (A). (C) Enlarged image of the area marked by a square in panel d in (A). Scale bar in B and C = 200 µm.

Figure 3

Expression of hGPR56 e1m-driven EGFP in transgenic marmoset embryo. (A) EGFP signals in the Dorsal view (a–c), ventral view (d–f), lateral view (g–i), and median view (j–l) of the whole brain of wild type (left panels) and transgenic marmoset (middle and right panels) at E95. Thalamus (Th), hypothalamus (Hy), midbrain (MB), cerebellum (Cb), medulla oblongata (MO), cerebral cortex (Cx), and olfactory bulb (OB) are shown. (B) Immunofluorescent staining of coronal sections from the anterior (a) to posterior (d) of the transgenic marmoset brain (E95) for EGFP. Lines on the schema indicate the position of each section. Cingulate gyrus (CG), early caudate (eCd), early putamen (ePu), hippocampus (HIP). Scale bars in A and B = 1 mm. (C) Coronal section of cerebral cortex of the transgenic marmoset brain (E95) stained for DNA (Hoechst) and EGFP. Cortical plate (CP), subplate (SP), IZ, oSVZ, iSVZ, and VZ are indicated. Scale bar = 200 µm. (D) Enlarged image of the area marked by a square in the panel of (B), with indication for Cd, ventricular zone of Cd (vCd), and subventricular zone of Cd (svCd).

Figure 4

hGPR56 e1m-EGFP expression in the cerebral cortex. (A) Immunofluorescent staining of coronal sections from the anterior (a) to posterior (e) of the transgenic marmoset brain at E113 for EGFP (green) and pan-GPR56 (magenta). Lines on the schema indicate the position of each section. Scale bar = 1 mm. (B) Coronal section of cerebral cortex of the transgenic marmoset brain at E113 stained for Hoechst (Blue), hGPR56 e1m-EGFP (green), and pan-GPR56 (magenta), CTIP2 or GABA (cyan). The section stained for CTIP2 is the one 50 µm anterior to the other sections. Cortical plate (CP) consisting of layers I to VI, SP, IZ, oSVZ, iSVZ, and VZ are indicated. Scale bar = 200 µm. (C) Distribution of GPR56 (magenta), hGPR56 e1m-EGFP (green), and CTIP2 or GABAs (cyan) positive cells in layer V of the cortical plate. Representative hGPR56 e1m-EGFP positive cells with (filled arrowheads) or without (open arrowheads) expression of CTIP2 or GABAs are marked. The panels furthest to the right display orthogonal views of a hGPR56 e1m-EGFP positive cell marked by the arrow. Orthogonal views of other hGPR56 e1m-EGFP positive cells are shown in Figure S5A. We used ZEN 2009 software (version: 6.0.0.303, Carl Zeiss, Oberkochen, Germany) to construct orthogonal views. Scale bar = 50 µm. (D) Ratio of hGPR56 e1m-EGFP positive cells in each CP layer of transgenic marmoset no. 1 and no. 2. (E) Ratio of CTIP2⁺ or GABA⁺ cells among the hGPR56 e1m-driven EGFP⁺ cells in layer V of transgenic marmosets no. 1 and no. 2. (F) Ratio of CTIP2⁺ or GABA⁺ cells among the pan-GPR56⁺ cells in layer V of transgenic marmosets no. 1 and no. 2.

Figure 5

Subtype marker expression in the hGPR56e1m-EGFP positive GABAergic interneurons. (A) Distribution of hGPR56e1m-EGFP and PV, SST or Calretinin in the cortical plate of the transgenic marmoset brain (E113). Scale bar = 1 mm. Enlarged images of the area marked by rectangles in top panels are shown in the lower panels. The dashed ellipses indicate the positive cells of each subtype marker of GABAergic neuron. Scale bars = 50 µm. (B) Ratio of PV⁺, CR⁺, or STT⁺ cells among the hGpr e1m-driven EGFP⁺ cells in the layer V of transgenic marmoset no. 1 and no. 2. (C) Ratio of PV⁺, CR⁺, or STT⁺ cells among the cells in the layer V of transgenic marmoset no. 1 and no. 2.