Loss of GABAergic neurons in the hippocampus and cerebral cortex of Engrailed-2 null mutant mice: implications for autism spectrum disorders

Abstract

The homeobox-containing transcription factor Engrailed-2 (En2) is involved in patterning and neuronal differentiation of the midbrain/hindbrain region, where it is prominently expressed. En2 mRNA is also expressed in the adult mouse hippocampus and cerebral cortex, indicating that it might also function in these brain areas. Genome-wide association studies revealed that En2 is a candidate gene for autism spectrum disorders (ASD), and mice devoid of its expression (En2(-/-) mice) display anatomical, behavioral and clinical "autistic-like" features. Since reduced GABAergic inhibition has been proposed as a possible pathogenic mechanism of ASD, we hypothesized that the phenotype of En2(-/-) mice might include defective GABAergic innervation in the forebrain. Here we show that the Engrailed proteins are present in postnatal GABAergic neurons of the mouse hippocampus and cerebral cortex, and adult En2(-/-) mice show reduced expression of GABAergic marker mRNAs in these areas. In addition, reduction in parvalbumin (PV), somatostatin (SOM) and neuropeptide Y (NPY) expressing interneurons is detected in the hippocampus and cerebral cortex of adult En2(-/-) mice. Our results raise the possibility of a link between altered function of En2, anatomical deficits of GABAergic forebrain neurons and the pathogenesis of ASD.

Keywords: ASD; CALB; CCK; DG; En2; Engrailed-2; GABA; GAD; Homeobox transcription factor; Inhibition; Interneuron; NPY; Neurodevelopmental disorder; PV; Parvalbumin; RT-PCR; SNP; SOM; VIP; autism spectrum disorders; calbindin 28kD; cholecystokin; dentate gyrus; glutamic acid decarboxylase; neuropeptide Y; parvalbumin; reverse transcription polymerase chain reaction; single-nucleotide polymorphism; somatostatin; vGAT; vGLUT; vasointestinal peptide; vesicular GABA transporter; vesicular glutamate transporter; γ-aminobutyric acid.

Figure 1. En1/2 proteins are expressed in the postnatal mouse hippocampus and somatosensory cortex

A,B) Triple immunostaing showing En- (red), PV- (green) and CALB- (blue) positive cells in the P28 mouse somatosensory cortex. C,D,E) Single-channel acquisitions of En, PV and CALB immunostains in deep cortical layer (detail of inset shown in A). F,G,H) Triple immunostaing showing En (red), PV (green) and CALB (blue) positive cells in the P28 mouse hippocampus. I,J,K) Single-channel acquisitions of En, PV and CALB immunostainings from the CA2-CA3 boundary (detail of inset shown in G). In all pictures, green-red arrowheads indicate PV-En double positive neurons, and blue-red arrowheads show CALB-En double positive neurons. Green and blue arrows represent PV and CALB neurons not expressing En. Abbreviations: En, Engrailed1/2; PV, parvalbumin; CALB, calbindin; DG, dentate gyrus; CA1-3, hippocampal pyramidal cell layer 1-3. Cortical layers are indicated in A). Scale bar: 400 μm (A), 700 μm (F,G); 95 μm (B-E); 160 μm (H-K).

Figure 2. Normal layering of the En2^−/− hippocampus

A) Representative NeuN immunostaining of the whole dorsal hippocampus from WT and En2^−/− mice. B) Subdivision of hippocampal layers used for morphometric analyses. C) Morphometric analysis of hippocampal layers in WT and En2^−/− mice. Layer thickess is plotted as % of dorsal hippocampus total thickness. Animal numbers and genotypes are as indicated. Abbreviations: DG, dentate gyrus; Lacun. Molec., stratum lacunosum moleculare. NeuN, pan-neuronal marker NeuN. Scale bars: 800 μm (A), 300 μm (B).

Figure 3. Reduced number of PV, NPY and SOM interneurons in the En2^−/− hilus

A) Representative pictures showing GAD-, PV-, NPY-, SOM- and CALB-positive interneurons in the hilus of adult WT and En2^−/− mice. B) GAD-positive hilar interneuron cell counts. Values are expressed as the mean number (± s.e.m) of positive cells per area (0.1 mm², see Material and Methods). C) Subtype-specific hilar interneuron cell counts. Values are reported as in B). Animal numbers and genotypes are as indicated. Asterisk indicates statistical significance (*p< 0.05, Student’s t-test, WT vs. En2^−/−). D) Relative mRNA expression level of glutamatergic and GABAergic markers, as obtained by quantitative RT-PCR performed on the whole hippocampus of adult WT and En2^−/− mice. Values are expressed as each marker/L41 comparative quantitation ratios (mean ± s.e.m of 3 replicates from pools of 7 animals per genotype; *p< 0.05, **p< 0.01, Student’s t-test, WT vs. En2^−/−). Abbreviations: vGLUT, vesicular glutamate transporter; vGAT, vesicular GABA transporter; GAD65/67, glutamic acid decarboxylase (65/67 kD isoform); PV, parvalbumin; CALB, calbindin; NPY, neuropeptide Y; SOM, somatostatin. Scale bar: 300 μm.

Figure 4. Normal layering of the En2^−/− somatosensory cortex

A) Representative NeuN immunostaining of the somatosensory cortex from adult WT and En2^−/− mice. B) Morphometric analysis of cortical in WT and En2^−/− mice. Layer thickess is plotted as % of total cortical thickness. Layers, animal numbers and genotypes are as indicated. Scale bar: 120 μm.

Figure 5. Reduced number of GABAergic interneurons in the En2^−/− somatosensory cortex

A) Representative in situ hybridizations showing GAD65/67 mRNA-positive neurons in the somatosensory cortex of adult WT and En2^−/− mice. B) GAD65/67 mRNA-positive interneuron cell counts. Values are expressed as the mean number (± s.e.m) of positive cells per area (0.12 mm², see Material and Methods). C) Representative immunostainings of PV, NPY and SOM interneurons in the somatosensory cortex of adult WT and En2^−/− mice. D) PV, NPY and SOM interneuron cell counts (values are reported as in B). E) Representative immunostainings of CALB (green) and GAD (red) positive neurons in the somatosensory cortex of adult WT and En2^−/− mice. F) Top: representative GAD (red arrowhead), CALB (green arrowhead) and GAD+CALB (white arrowhead) interneurons in layers V-VI of the En2^−/− somatosensory cortex. Bottom: cell counts of CALB/GAD double-positive interneurons in the somatosensory cortex of adult WT and En2^−/− mice (values are reported as in B). G) Relative expression of glutamatergic and GABAergic markers mRNAs in the somatosensory cortex of adult WT and En2^−/− mice (quantitative RT-PCR). Values are plotted as the mean ± s.e.m of 3 replicates from pools of 7 animals per genotype. In all pictures, abbreviations are as in Fig. 3 and animal numbers, genotypes and cortical layers are as indicated. In all graphs, asterisks indicate statistical significance (*p< 0.05, ***p< 0.001, Student’s t-test, WT vs. En2^−/−). Scale bars: 200 μm (A), 85 μm (C,E), 20 μm (F).