Agenesis of the Corpus Callosum Due to Defective Glial Wedge Formation in Lhx2 Mutant Mice - PubMed (original) (raw)

Agenesis of the Corpus Callosum Due to Defective Glial Wedge Formation in Lhx2 Mutant Mice

Gregory A Chinn et al. Cereb Cortex. 2015 Sep.

Abstract

Establishment of the corpus callosum involves coordination between callosal projection neurons and multiple midline structures, including the glial wedge (GW) rostrally and hippocampal commissure caudally. GW defects have been associated with agenesis of the corpus callosum (ACC). Here we show that conditional Lhx2 inactivation in cortical radial glia using Emx1-Cre or Nestin-Cre drivers results in ACC. The ACC phenotype was characterized by aberrant ventrally projecting callosal axons rather than Probst bundles, and was 100% penetrant on 2 different mouse strain backgrounds. Lhx2 inactivation in postmitotic cortical neurons using Nex-Cre mice did not result in ACC, suggesting that the mutant phenotype was not autonomous to the callosal projection neurons. Instead, ACC was associated with an absent hippocampal commissure and a markedly reduced to absent GW. Expression studies demonstrated strong Lhx2 expression in the normal GW and in its radial glial progenitors, with absence of Lhx2 resulting in normal Emx1 and Sox2 expression, but premature exit from the cell cycle based on EdU-Ki67 double labeling. These studies define essential roles for Lhx2 in GW, hippocampal commissure, and corpus callosum formation, and suggest that defects in radial GW progenitors can give rise to ACC.

Keywords: Cre-lox; Lhx2; agenesis of the corpus callosum; glial wedge; mouse.

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Figures

Figure 1.

Absence of corpus callosum and hippocampal commissure, but preserved anterior commissure, in Emx1/Lhx2 and Nestin/Lhx2 mutant adults. H&E histology, coronal sections; dashed boxes in A–A″ are magnified in B–B″. (A,B) Normal corpus callosum in an Emx1-Cre-positive control littermate. (A′–B′,A″–B″) Emx1/Lhx2 and Nestin/Lhx2 mutants lack a corpus callosum, and misrouted ventrally oriented axons are present in the septum (black arrowheads). Probst bundles are not seen. (C–C″) In contrast to an Emx1-Cre-positive control, Emx1/Lhx2 and Nestin/Lhx2 mutants lack a hippocampal commissure. (D–D″) Anterior commissure is intact in Emx1-Cre control, Emx1/Lhx2 mutant, and Nestin/Lhx2 mutant mice. Abbreviations: CC, corpus callosum; HC, hippocampal commissure; AC, anterior commissure. Scale bars: 200 μm.

Figure 2.

Ventrally oriented callosal axons in Emx1/Lhx2 mutant mice. DiI and in utero electroporation studies, coronal sections. (A–D) Callosal axons cross the midline in Emx1/Lhx2 control adults following neocortical placement of DiI crystals (A,B) or in control E19.5 animals following GFP electroporation at E14.5 (C,D). (A′–D′) DiI- or GFP labeled callosal axons (white arrowheads) in Emx1/Lhx2 mutant littermates do not cross the midline and are ventrally oriented. Rostrocaudally oriented axons typical of Probst bundles are not apparent. White asterisks designate the end of visible DiI labeling in callosal axons. Scale bars: 100 μm.

Figure 3.

Normal specification of callosal and corticospinal projection neurons in Emx1/Lhx2 and Nestin/Lhx2 mice. (A–D,A′–D′) Satb2 expression is widespread in both Emx1/Lhx2 control and mutant cortex at E18.5. In the same sections, Ctip2 expression is confined to lower cortical layers. (D,D′) Satb2 and Ctip2 exhibit mutually exclusive staining in both control and mutant neocortical neurons, suggesting normal specification of callosal and corticospinal neurons, respectively. (E,E′) TuJ1 staining of Nestin/Lhx2 at E16.5 highlight the normal and abnormal tracts of callosal projection neurons in controls and mutants. Scale bars: 100 μm.

Figure 4.

Presence of corpus callosum in Nex/Lhx2 mice. (A,A′) LacZ histochemistry, E18.5 Nex/R26R coronal sections. Widespread lacZ expression occurs in the cortical plate, but not in the VZ, glial wedge, or other forebrain regions. Xgal staining of the corpus callosum confirms Nex-Cre-mediated recombination in callosal neurons. Boxed region in A is magnified in A′. (B–D,B′–D′) Lhx2 IHC, E14.5 Nex/Lhx2 coronal sections. Lhx2 expression normally occurs in all regions of the cortical VZ and cortical plate (CP), including lateral neocortex (C) and rostromedial neocortex (D). In Nex/Lhx2 mutants, little-to-no Lhx2 expression is detected in all CP regions, while VZ expression persists (B′–D′). (E,E′) H&E stains, P7 Nex/Lhx2 coronal sections. A corpus callosum of similar caliber is present in both control and mutant littermates. Abbreviations: VZ, ventricular zone; IZ, intermediate zone; CP, cortical plate. Scale bars: 200 μm.

Figure 5.

GW deficits in Emx1/Lhx2 and Nestin/Lhx2 mutant mice. H&E histology and GFAP IHC, coronal sections; boxed regions are magnified in panels immediately below each low-power image. (A–C) At E16.5–18.5, the GW in control littermates (black arrowheads) is a cell-dense, wedge-shaped structure extending from the ventricle and situated between rostromedial cortex and septum. (A′–C′) Little to no GW is apparent in E16.5–18.5 Emx1/Lhx2 mutant embryos. The GW region contains eosinophilic cell-sparse zones corresponding to the misrouted callosal axons. (D,D′) GFAP-positive stellate astrocytes seen in the normal GW of E18.5 control animals (arrowheads in D) are absent from an Emx1/Lhx2 mutant (_D_′). (E,E′) E16.5 Nestin/Lhx2 ISH for Bmp6 in the control faintly marks the GW (black arrowheads) as well as a medial cell population dorsal to the corpus callosum (asterisks). No expression is detected in the mutant GW, while medial Bmp6 expression is maintained. Scale bars: 100 μm.

Figure 6.

Lhx2 and Emx1-Cre expression in the developing GW. (A–C,A′–C′) Lhx2 IHC (green) and phalloidin staining (red), E15.5 coronal sections. Lhx2 expression spans the medial cortical wall, including the GW and GW VZ (white arrowheads). Lhx2 is also expressed in the septal VZ, but less so in the septal mantle zone. Phalloidin staining also demarcates the cortical-septal boundary and GW region. (D,D′) Lhx2 in situ hybridization, P4 coronal section. Lhx2 expression in the GW is maintained into the postnatal period. (E–E′,F–F′) LacZ histochemistry, E18.5 and P4 Emx1/Lhx2 coronal sections. Expression of the Emx1-Cre allele, which contains a lacZ reporter, is expressed in the GW VZ and GW proper of control littermates(E,F). In Emx1/Lhx2 mutants, far fewer Xgal-stained cells are seen in the GW region (E′,F′). (G–G′,H–H′) Sox2 IHC E12.5 and E16.5 Emx1/Lhx2 coronal sections. At both stages, Sox2 expression persists in GW progenitors of both Emx1-Cre-positive littermate controls (G,H) and Emx/Lhx2 mutants (G′–H′). Abbreviations: VZ, ventricular zone. Scale bars: 100 μm.

Figure 7.

Impaired GW genesis in Emx1/Lhx2 and Nestin/Lhx2 mutant mice. EdU birthdating studies, E18 coronal sections. (A–C) EdU administration at E14 (A,B) or E16 (C) results in prominent EdU labeling (green) of the GW in control littermates. (A′–C′) Emx1/Lhx2 and Nestin/Lhx2 mutants show little-to-no EdU labeling in the GW. Scale bar: 100 μm.

Figure 8.

Abnormal radial GW progenitors in Emx1/Lhx2 mice. EdU studies, coronal sections. Red bars approximate the GW region. (_A,A_′) Following E11.5 administration, EdU label is abnormally retained in the GW VZ of an E12.5 Emx1/Lhx2 mutant (A′) compared with a control littermate (A). (B–C,B′–C′) Abnormal EdU retention in the GW VZ persists at E14.5 in mutants compared with littermate controls following E11.5 administration. (D–E,D′–E′) Acute administration (9 h prior to sacrifice) reveals far fewer EdU-labeled cells in the GW VZ of mutants compared with littermate controls. (F,G) EdU/Ki67 labeling reveals Ki67-positive and -negative subpopulations of EdU-labeled cells in both control and mutant E14.5 GW VZ/SVZ. (F′,F″) Examples of Ki67-positive and -negative EdU-labeled cells, respectively, in control VZ/SVZ. (G′,G″) Examples of Ki67-positive and -negative EdU-labeled cells, respectively, in mutant VZ/SVZ. (H) Quantification of EdU-positive cells in the GW VZ/SVZ reveals a statistically significant increase in mutants compared with littermate controls indicated by asterisks (5.13 ± 0.28 vs. 1.16 ± 0.17, P = 0.0003, n = 3). (I) Quantification of the Ki67-positive fractions of EdU-labeled cells in the GW VZ/SVZ reveals a significant decrease in mutant embryos compared with control littermates (28.29 ± 0.53 vs. 82.74 ± 3.90, P = 0.002, n = 3). Scale bars: A_–_E,F,G, 100 μm; F′,F″,G′,G″, 10 μm.

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Agenesis of the Corpus Callosum Due to Defective Glial Wedge Formation in Lhx2 Mutant Mice - PubMed (original) (raw)