Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members

Abstract

The Alzheimer's disease beta-amyloid precursor protein (APP) is a member of a larger gene family that includes the amyloid precursor-like proteins, termed APLP1 and APLP2. We previously documented that APLP2-/-APLP1-/- and APLP2-/-APP-/- mice die postnatally, while APLP1-/-APP-/- mice and single mutants were viable. We now report that mice lacking all three APP/APLP family members survive through embryonic development, and die shortly after birth. In contrast to double-mutant animals with perinatal lethality, 81% of triple mutants showed cranial abnormalities. In 68% of triple mutants, we observed cortical dysplasias characterized by focal ectopic neuroblasts that had migrated through the basal lamina and pial membrane, a phenotype that resembles human type II lissencephaly. Moreover, at E18.5 triple mutants showed a partial loss of cortical Cajal Retzius (CR) cells, suggesting that APP/APLPs play a crucial role in the survival of CR cells and neuronal adhesion. Collectively, our data reveal an essential role for APP family members in normal brain development and early postnatal survival.

Figure 1

Cortical dysplasia in APP^−/−APLP1^−/−APLP2^−/− triple knockout mice of various ages. (A, B) Frontal sections (cresyl violet staining) of a triple knockout mouse (T) at E18.5, exhibiting a prominent protrusion (P) of the right hemispheric cortical plate. The occurrence of ectopias was restricted to dorsal cortical areas as indicated by lines in (A). (B) Upon higher magnification (boxed area in A), it becomes apparent that ectopic neurons completely disrupt the subplate (SP) and cortical plate (CP); the cells have migrated into and beyond the MZ. No alterations were observed in the ventricular zone (not shown) (C, D) Depict low- and high-power images of a littermate control (C, genotype: APP^−/−APLP1^+/−APLP2^−/−). (F) Example of an E17.5 triple knockout brain (T) showing two smaller cerebral protrusions (P). Arrowheads mark cells invading the subarachnoidal space (s). (E) Example of an E14.5 triple knockout exhibiting a small protrusion in comparison to a littermate control (C, genotype: APP^−/−APLP1^−/−APLP2^+/−) depicted in (G). (H) Example of ectopia in a triple knockout embryo at E17.5. Note that a complete disruption of cortical layers is typically found within the center of dysplasias (B, H). In more lateral aspects (sections), the layering of deeper cortical structures appears much less affected (see, for example, F). Scale bars=500 μm (A, C), 100 μm (B, D, F, H), 50 μm (E, G).

Figure 2

Immunohistochemical characterization of ectopias in APP^−/−APLP1^−/−APLP2^−/− triple knockout mice at E18.5. (A, B) High-power views of cresyl violet-stained coronal sections of control (A) and (B) triple knockout cortex. (B) Shows a high power view of Figure 1B, depicting the border of a neuronal outgrowth with a well-defined MZ on the left and clustered ectopic neurons on the right (P, protrusion). (C, D) Staining of control (C) and triple knockout (D) sections for reticulin, a marker for collagen fibers in the basal lamina (BL) and pial layer revealed that the BL (arrow) was severely disrupted at the protrusion. Arrowheads indicate reticulin-stained thread-like abnormal structures. (E, F) CR cells in control (E) and triple mutant (F) sections stained for reelin (green, arrows). In controls and adjacent to ectopias, CR cells were properly located at the leptomeningeal side of the MZ. Note that, within ectopias, positioning of CR cells was severely disordered (as indicated by arrows) (G, H) Anti-laminin staining (red) of control (G) and triple mutant (H) sections. Triple mutants revealed normal laminin expression along the BL, which was, however, abrogated at the border neighboring the outgrowths (arrow). Aberrant punctate laminin staining is indicated by arrowheads. (K, L, O, P) Staining of radial glia (using an antibody against EAAT1, green) in the cerebral cortex of a control (K, O) and triple knockout. (L, P) Nuclei were stained with DAPI (see overlay depicted in P). (K, O) In control mice, radial glial fibers were found to extend through the cortical plate and MZ to the meningeal cell layer. (L) Within the protrusion, very little EAAT1 staining was detectable and fibers terminated prematurely. However, in unaffected regions of triple mutant cortex (inset in L depicts a high-power magnification), radial glial foot processes terminated normally at the pial–glial interface. (I, J, M, N) Immunostaining for MAPII (green) and counterstaining of nuclei with DAPI (blue) of control (I, M) and triple mutant (J, N) sections. Note that, within ectopias, MAPII staining is grossly disorganized. MZ, marginal zone; CP, cortical plate; scale bars=50 μm for all panels, except for the 15 μm scale bar within the inset in (L). Genotype of littermate controls: APP^−/−APLP1^+/−APLP2^−/−.

Figure 3

Neuronal migration in the cerebral cortex is unaltered in triple mutants. (A–F) BrdU birthdating analysis. Pregnant mice were injected with BrdU either at E12.5 or at E15.5 and the distribution of BrdU⁺ neurons (green) was determined at E18.5 and E19.5, respectively. (A, C) Sections of littermate controls (C) and (B, D) triple mutants (T) injected at E12.5 (A, B), or E15.5 (C, D). Panels on the right show a quantification of BrdU⁺ cells at various distances from the MZ. BrdU⁺ cells were counted in six stripes (consisting of five squares of 50 × 50 μm²) positioned underneath the MZ along the vertical axis. Note that statistical analysis (Student's t-test) did nor reveal any significant changes (P>0.05) in the distribution of BrdU⁺ neurons between triple knockouts (black bars) and littermate controls (open bars). The distribution of neurons labeled at E12.5 peaks within deeper layers of the cortical plate, whereas the majority of neurons labeled at E15.5 were found underneath the MZ. Values represent mean±s.d. of the percentage BrdU⁺ cells/stripe area relative to all positive cells from all six stripes obtained from four to five animals. (F) A high proportion of E12.5-injected BrdU⁺ cells are found throughout the protrusion of a triple knockout embryo sectioned at E18.5. (E) Unaffected littermate control for comparison. Genotypes of littermate controls: APP^−/−APLP1^−/−APLP2^+/−. Scale bars=50 μm.

Figure 4

Alterations in the MZ of triple knockout mice. (A, B) Appearance of the MZ in HE-stained sections of (A) littermate control and (B) triple knockout cortices at E16.5. Note the prominent reduction in the number of nuclei in the MZ of triple mutants. (D, E) Immunohistochemistry for reelin (red) revealed a considerable reduction of CR cells in triple knockouts (E) as compared to controls (D) at E18.5. (C) The number of reelin-positive neurons within a 1200 μm wide MZ strip of the parietal cortex of E18.5 embryos was determined for both hemispheres on 8-μm-thick frozen frontal cortical sections. Values represent average cell counts±s.d. from n=8 stripes of four triple mutants (T, filled bar) and n=10 stripes of five littermate controls (C, open bar). ^*P<0.05, Student's t-test. (F–K) Frontal sections of E18.5 cortices from littermate control (F, I) and triple mutants (G, H, J, K) stained with antibodies against CSPG (F–H, red) or mDab1 (I–K, red). Cell nuclei were stained with DAPI (blue). The expression of CSPG and mDab1 within the MZ of triple knockouts (G, J) appeared unaffected in areas lacking protrusions. However, within protrusions (H, K), the normal pattern of expression of CSPG (H) and mDab1 (K) was interrupted and appeared disorganized. Scale bars=30 μm. Genotypes of littermate controls: APP^−/−APLP1^−/−APLP2^+/− (A, D), APP^−/−APLP1^+/+APLP2^+/+ (F), APP^−/−APLP1^+/+APLP2^+/− (I).