Agenesis of the corpus callosum in Nogo receptor deficient mice

Abstract

The corpus callosum (CC) is the largest fiber tract in the mammalian brain, linking the bilateral cerebral hemispheres. CC development depends on the proper balance of axon growth cone attractive and repellent cues leading axons to the midline and then directing them to the contralateral hemisphere. Imbalance of these cues results in CC agenesis or dysgenesis. Nogo receptors (NgR1, NgR2, and NgR3) are growth cone directive molecules known for inhibiting axon regeneration after injury. We report that mice lacking Nogo receptors (NgR123-null mice) display complete CC agenesis due to axon misdirection evidenced by ectopic axons including cortical Probst bundles. Because glia and glial-derived growth cone repellent factors (especially the diffusible factor Slit2) are required for CC development, their distribution was studied. Compared with wild-type mice, NgR123-null mice had a sharp increase in the glial marker glial fibrillary acidic protein (GFAP) and in Slit2 at the glial wedge and indusium griseum, midline structures required for CC formation. NgR123-null mice displayed reduced motor coordination and hyperactivity. These data are consistent with the hypotheses that Nogo receptors are membrane-bound growth cone repellent factors required for migration of axons across the midline at the CC, and that their absence results directly or indirectly in midline gliosis, increased Slit2, and complete CC agenesis. J. Comp. Neurol. 525:291-301, 2017. © 2016 Wiley Periodicals, Inc.

Keywords: NgR; Probst bundle; Slit2; axon; corpus callosum; glia.

Figure 1

Neuroanatomy of NgR123-null mice. A–D: Nissl-stained coronal brain sections from 12-week old wild type (A,C) and NgR123-null (B,D) mice at two different levels. Callosal agenesis was found in 16/16 NgR123-null and 0/10 wild type (WT) mice. Scale bar = 1 mm. E–F: NeuN immunostained caudal midline structures in a 12-week old wild type (E) and NgR123-null (F) mouse. CC, Corpus callosum; FC, Fasciola cinereum. Arrows highlight CC agenesis. Scale bar = 200 µm. G–N: CC agenesis and Probst bundle formation in NgR123-null mice. Coronal brain sections from 12-week old wild type (G,I,K,M) and NgR123-null (H,J,L,N) mice were stained with Eriochrome/Eosin (G–J) and Luxol Fast Blue (K–N). Insets are magnified images at the midline. Arrows indicate loss of the CC and arrowheads indicate Probst bundles. Scale bar is 1 mm for the larger image and 250 µm for the insets.

Figure 2

Ectopic white matter tracts in the cingulate cortex of NgR123-null mice. Myelinated fibers in coronal sections from brains of 12-week old wild type (A,C,E) and NgR123-null mice (B,D,F) were stained with Eriochrome/Eosin (A,B) or immunostained with anti-MBP (C,D) or anti-CNPase (E,F). Synaptic staining of wild type (G) and NgR123-null (H) coronal sections used anti-synaptophysin antibody. The dotted line indicates loss of synapses in the cingulate cortex of NgR123-null mice. Boxed areas at the cortical midline (top row) are presented at higher magnification below each complete coronal section (middle row), with selected areas corresponding to regions of ectopic fibers at higher power (bottom row). Scale bar = 50 µm (bottom row), 280 µm (middle row) and 1.9 mm (top row).

Figure 3

Immunoblots of neuronal, synaptic and myelin markers in extracts of midline structures from wild type and NgR123-null mice. Homogenates of midline structures dissected from 3 mice of each group were resolved by SDS-PAGE, transferred, and blotted with specific antibodies for neurons (anti-βIII-tubulin), synapses (anti-synaptophysin), myelin (anti-CNPase and anti-MBP) and a loading control protein (anti-GAPDH). Densitometry of each band was normalized to GAPDH in the same sample. Normalized data (relative to GAPDH) were compared by Student’s t-test (*, p < 0.05; **, p < 0.01). Data points are presented relative to the wild type average, with each data point for wild type (open symbols) and NgR123-null (grey symbols) presented. Averages for each genotype are denoted with a plus sign (+).

Figure 4

Callosal agenesis during postnatal development in NgR123-null mice. Coronal sections from wild type (A,C,E,G,I) and NgR123-null (B,D,F,H,J) mice were stained with Eriochrome/Eosin. Brains from mice of the following post-natal ages are shown: 1 week (A,B), 2 weeks (C,D), 4 weeks (E,F), 12 weeks (G,H) and 1 year (I,J). Midline structures (right column) of NgR123-null mice at higher power are presented to the right of the corresponding complete coronal sections. Arrows indicate disconnected corpus callosum and arrowheads indicate Probst bundles. The scale bar is 1 mm for complete coronal sections and 200 µm for midline images.

Figure 5

Glial marker and Slit2 expression in midline structures of wild type and NgR123-null mice. A–D: Coronal sections of brains from 12-week (A,B) and 1-year old (C,D) wild type (A,C) and NgR123-null (B,D) mice were immunostained for GFAP. The cingulate cortex (Cg, one of two bilateral labeled), CC, glial wedge (gw, one of two bilateral labeled) and indusium griseum (Ig) are labeled. E: Average GFAP-immunostained midline and hippocampal binary pixel areas from 5 animals from each group at each age. Values were normalized to the 12-week old wild type for presentation. Pairwise comparisons (Student t-test) revealed a significant increase in GFAP immunostaining only in the midline of 12-week old NgR123-null mice (**, p < 0.01). F: Failure of midline fusion in NgR123-null mice. Relative areas bounded by the bilateral cingulate cortices and the indusium griseum of wild type and NgR123-null mice (5 animals each) are presented (**, p < 0.01). G–N: DAPI, GFAP and Slit2 immunostaining of midline cortical regions in coronal brain sections from 12-week old wild type (G,I,K,M) and NgR123-null (H,J,L,N) mice. Staining by DAPI (blue nuclei, G,H), anti-GFAP (green, I,J), anti-Slit2 (magenta, K,L), and anti-GFAP/anti-Slit2 overlay (M,N) are shown.

Figure 6

Glial markers in midline extracts from wild type and NgR123-null mice. (A) Homogenates of cerebral midline tissue from wild type and NgR123-null mice (three mice each) were resolved by SDS-PAGE, transferred, and blotted with specific antibodies for astrocytes (anti-GFAP), Slit2 and ligands for NgRs including Nogo-A, OMgp and MAG. (B,C) Densitometry of each band was normalized to the loading control protein GAPDH and is presented by fold induction based on wild type. Data are presented as mean ± SE. Pairwise differences between mutant and wild type (Student t-test): *, p<0.05. Data points are presented relative to the wild type average, with each data point for wild type (open symbols) and NgR123-null (grey symbols) presented. Averages for each genotype are denoted with a plus sign (+).

Figure 7

Behaviors of NgR123-null mice. (A) Motor coordination was evaluated by Rotarod at 12 weeks and 1 year of age. Duration time on the drum was measured starting at 4 rpm and accelerating an additional 4 rpm every 30 sec. (B) Open field activity of 1 year-old mice was evaluated using infrared beam breaks in an open field frame. Total beam breaks in 120 sec were averaged for 5 trials for each mouse. Data are presented as mean ± SE. Pairwise differences were determined by Student t-test (n=8; *, p<0.05; **, p<0.01).