Defective laminin organization in FE65^−/−; FE65L1^−/− meningeal fibroblasts. (A) Primary meningeal fibroblasts from WT and FE65^−/−; FE65L1^−/− mice were double stained with a laminin antibody and phalloidin-AlexaFluor 488 to detect F-actin. Fibrillar and punctate laminin is observed at the periphery in WT meningeal fibroblasts, whereas it is absent from FE65^−/−; FE65L1^−/− meningeal fibroblasts. Scale bar, 20 μm. (B) Staining with the lectin RCA-1 shows no difference in the thickness of meninges for WT and FE65^−/−; FE65L1^−/− E18.5 mouse brains.

Aberrant Tag-1 corticofugal and L1-thalamocortical fiber projections in FE65^−/−; FE65L1^−/− mice. IHC staining of Tag-1 (A, B) and L1 (C, D) in coronal cryostat sections of E16.5 WT (A, C) and FE65^−/−; FE65L1^−/− (B, D) mouse brains. There is a reduction in Tag-1-positive fibers exiting the cortex in FE65^−/− FE65L1^−/− mice (B) compared to WT controls (A). Fewer L1-positive fibers enter the cortex from the caudate putamen (CaP) and are ectopically localized in the developing cortex of FE65^−/−; FE65L1^−/− mice (D) compared to the organized projections of L1-positive fibers in the WT control (C). Arrows identify L1-positive ectopic fibers that have reached the marginal zone.

Cortical abnormalities in FE65^−/−; FE65L1^−/− mice. H&E-stained coronal sections of E18.5 (A–D) and E12.5 (E–F) mouse cortices. Control littermate FE65^−/−; FE65L1^+/− (A, C) and E12.5 WT (E) control sections are compared to FE65^−/−; FE65L1^−/− E18.5 (B, D) and E12.5 sections (F). Arrowheads point to ectopic cell nodules on the surface of the E18.5 cortex in the FE65^−/−; FE65L1^−/− brain (B) and arrows indicate ectopic neurons that have invaded the marginal zone and broken through the pial membrane of E18.5 and E12.5 cortex (D, F). The cranium is present in E12.5 sections. Scale bar, 100 μm (B–F). MZ, marginal zone; CP, cortical plate; IZ, intermediate zone; VZ, ventricular zone; VP, ventral pallium; GE, ganglionic eminence.

APP and Aβ levels in FE65^−/−; FE65L1^−/− brains. (A) IHC staining for APP (A8717) shows expression throughout the cortical plate in WT and FE65^−/−; FE65L1^−/− E18.5 brains and in heterotopic cells. (B) APP and APLP2 steady-state levels are unchanged in FE65^−/−; FE65L1^−/− adult mouse brains compared to WT. Western blot analyses using 22C11 and anti-β-tubulin (reprobe) on two mouse brain diethylamine extracts per genotype. Bar chart shows average APP steady-state levels that were normalized to β-tubulin (as load control), with standard error bars for WT (n=8), FE65^−/− (n=7), FE65L1^−/− (n=9), FE65^−/−; FE65L1^−/− (n=9) mouse brains. No significant difference was observed between genotypes. (C) Aβx-40 and Aβx-42 levels in diethylamine brain extracts of WT (n=5F, 7M), FE65^−/−(n=7F, 4M), FE65L1^−/− (n=10F, 4M) and FE65^−/−; FE65L1^−/− (n=6F, 6M) 14- to 21-week-old mice. Data have been normalized to the mean of the WT mice. Aβx-42 levels are significantly reduced in FE65^−/−; FE65L1^−/− males compared to WT. t-Test ^*P<0.05; ^**P<0.005.

Reelin-positive neurons, reelin and ECM components in the developing cortical plate of FE65^−/−; FE65L1^−/− mice. IHC staining of coronal paraffin sections from E18.5 (A, B), E14.5 (C–H) and E12.5 (I–N) control (A, C, E, G, I, K, M) and FE65^−/−; FE65L1^−/− (B, D, F, H, J, L, N) mouse brains. In WT E18.5 and E14.5 cortices (A, C), G10 staining shows reelin-positive neurons and secreted reelin in the marginal zone. In the FE65^−/−; FE65L1^−/− mice (B, D), reelin-positive cells are displaced by ectopic neurons. In WT cortices, CSPGs (CS56 antibody) are present in the subplate and marginal zone (E), the latter being disrupted by heterotopic neurons in FE65^−/−; FE65L1^−/− mice (F). Laminin staining reveals blood vessels and the cortical basement membrane (G), which is discontinuous when neurons invade the marginal zone of FE65^−/−; FE65L1^−/− mouse brains (H). G10 staining shows reelin-positive cells in the ventral pallium of E12.5 WT brains (I), which have overmigrated in the E12.5 FE65^−/−; FE65L1^−/− mouse brain (J). CSPG (K, L) and laminin (M, N) staining in the ventral pallium of E12.5 brains is disrupted at the pial membrane by heterotopic cells in the FE65^−/−; FE65L1^−/−brains. Arrowheads in panels B, D, F, H, L and N show the breaks in the pial membrane associated with heterotopias. Arrows in panels A and B point to reelin-positive cells. MZ, marginal zone; CP, cortical plate; SP, subplate; IZ, intermediate zone.

Abnormal axonal trajectories in FE65^−/−; FE65L1^−/− mouse cortex. Silver-stained coronal sections of 10-week-old WT (A, C), 12-week-old FE65^−/−; FE65L1^−/− (B, D) and 9-week-old (E) FE65^−/−; FE65L1^−/− mouse brain sections. An intact corpus callosum, well-developed fimbria and mossy fiber axons emanating from dentate granule cells are observed in the WT mouse brain (A). In the FE65^−/−; FE65L1^−/− mice, cortical projection neurons fail to cross the midline, forming Probst bundles, and fimbria are reduced in size and medially displaced (B). Higher magnification (boxed in A, B) reveals the absence of the infrapyramidal mossy fibers in FE65^−/−; FE65L1^−/− hippocampi (D) compared to WT (C). Aberrant fiber bundles are present in the cortical plate of adult (E) and E18.5 FE65^−/−; FE65L1^−/− mice (F). E18.5 brain slices were stained with Tuj1 (red) and counterstained with DAPI (blue) (F). Arrows indicate the position of fiber bundles and arrowheads indicate breaks in the marginal zone where DAPI-positive cells have invaded and migrated into the subarachnoid space. cc, corpus callosum; dg, dentate gyrus; f, fimbria; mf, mossy fibers; spmf, suprapyramidal mossy fibers; ipmf, infrapyramidal mossy fibers; P, Probst bundle. Scale bar, 250 μm (A).