Lis1-Nde1-dependent neuronal fate control determines cerebral cortical size and lamination

Abstract

Neurons in the cerebral cortex originate predominantly from asymmetrical divisions of polarized radial glial or neuroepithelial cells. Fate control of neural progenitors through regulating cell division asymmetry determines the final cortical neuronal number and organization. Haploinsufficiency of human LIS1 results in type I lissencephaly (smooth brain) with severely reduced surface area and laminar organization of the cerebral cortex. Here we show that LIS1 and its binding protein Nde1 (mNudE) regulate the fate of radial glial progenitors collaboratively. Mice with an allelic series of Lis1 and Nde1 double mutations displayed a striking dose-dependent size reduction and de-lamination of the cerebral cortex. The neocortex of the Lis1-Nde1 double mutant mice showed over 80% reduction in surface area and inverted neuronal layers. Dramatically increased neuronal differentiation at the onset of corticogenesis in the mutant led to overproduction and abnormal development of earliest-born preplate neurons and Cajal-Retzius cells at the expense of progenitors. While both Lis1 and Nde1 are known to regulate the mitotic spindle orientation, only a moderate alteration in mitotic cleavage orientation was detected in the Lis1-Nde1 double deficient progenitors. Instead, a striking change in the morphology of metaphase progenitors with reduced apical attachment to the ventricular surface and weakened lateral contacts to neighboring cells appear to hinder the accurate control of cell division asymmetry and underlie the dramatically increased neuronal differentiation. Our data suggest that maintaining the shape and cell-cell interactions of radial glial neuroepithelial progenitors by the Lis1-Nde1 complex is essential for their self renewal during the early phase of corticogenesis.

Figure 1.

Lis1 and Nde1 function together in cerebral cortical development. (A) Brains of adult mice with Nde1^+/−, Lis1^+/−, Nde1^+/−Lis1^+/− and Nde1^−/− mutations. Compared to Nde1 and Lis1 single heterozygous mutants, the brains of double heterozygous mutants (Nde1^+/−Lis1^+/−) showed an approximately 20–25% reduction by weight (n=13, P < 0.001), resembling that of Nde1^−/− mutants. (B, C) Histological and Immunohistological analyses suggested that the brain of the Nde1^+/−Lis1^+/− mutant was grossly normal, except for a significant thinning of the cortical layers II/III indicated by Cux1 immunoreactivity and blurred cortical layer boundaries. E, Nde1; L, Lis1. Bar: 200 µm.

Figure 2.

The Lis1–Nde1 complex is essential for determining cerebral cortical size, shape and lamina structures. (A) Dying at birth, the brains of Nde1^−/−Lis1^+/− mutant mice were dramatically reduced (arrows). A more pronounced size reduction of the cerebral hemispheres was observed. (B) Histological analysis showed that the neocortex of the Nde1^−/−Lis1^+/− mutant was severely disorganized, lacked the normal MZ and any other cortical layers. (C) Immunohistological analyses with layer-specific markers showed that in the Nde1^−/−Lis1^+/− cortex, cells belonging to superficial and middle cortical layers (marked by Cux1 and Foxp1 immunoreactivity, respectively) were greatly reduced, positioned randomly and often formed heterotopia in deeper cortical regions. In contrast, deep layer maker Tbr1 highlighted cells in the superficial cortex, suggesting grossly inverted cortical layers in the mutant. E, Nde1; L, Lis1. Bar: 200 µm (D) Rostral to caudal length (L1), medial to lateral length (L2) and cortical thickness (V) of Nde1^−/−Lis1^+/− cortex were measure and compared with those of the Nde1^+/− control at P0. The mutant cortex was less than 40% of the controls in L1 and L2, but only reduced by 20% in thickness (V).

Figure 3.

Reduction of VZ progenitors at the onset of corticogenesis by Lis1–Nde1 double deficiency. (A) The size of telencephalic vesicles (indicated by dashed blue circles) of the Nde1^−/−Lis1^+/− mutant appeared close to normal before E11, but was significantly smaller by E13 compared to that of their littermates. (B) Correlated with the thinning of cerebral cortex at E12.5, the thickness of cortical VZ and the number of S-phase neural progenitors examined by BrdU transient labeling (in red), was decreased significantly in the Nde1^−/−Lis1^+/− mutant. P < 0.001. E, Nde1; L, Lis1. Bar: 100 µm. (C) Reduced neural progenitors in the Nde1^−/−Lis1^+/− mutant cortex was indicated by reduced immunostaining of the glutamate transporter GLAST (in red), a marker of radial glial progenitors. Bar: 100 µm. (D) Substantial cell death was detected in the cerebral cortex of the Nde1^−/−Lis1^+/− mutant by TUNEL staining (in green). In contrast, very few TUNEL positive cells were detected in the Nde1^+/− and Nde1^+/−Lis1^+/− controls. Bar: 100 µm. (E) Majority (over 80%) of TUNEL positive cells (in green) in the Nde1^−/−Lis1^+/− mutant were newborn post mitotic neurons in the intermediate zone (IZ) and the cortical plate (CP) and expressed DCX (in red). Bar: 100 µm. (F) Cell-cycle exit profiles of Nde1^−/−Lis1^+/−progenitors. Pregnant females were given single does of BrdU at E12 and were analyzed 18 h later. Brain sections were stained with antibody to BrdU (in green) and to Ki67 (in red). Cells that exit the cell cycle are counted as those that are positive for BrdU but negative for Ki67, and presented as the percentage of total BrdU positive cells. Approximately 67% of cells labeled by BrdU at E12 became non-progenitor cells in the Nde1^−/−Lis1^+/− cortex by E13, while only 25–28% of the Nde1^+/−Lis1^+/− or Lis1^+/− progenitors left the cell cycle (P<0.001). Bar: 100 µm.

Figure 4.

Overproduction of PP neurons and lack of preplate splitting in the Nde1^−/− Lis1^+/− cortex. (A) Preplate neurons were labeled by CSPG antibody in red and cortical plate neurons were labeled with DCX antibody in green. A significant increase in CSPG was detected throughout the entire Nde1^+/−Lis1^+/− cortex before its size was reduced at E11.5. (B) The cortex of Nde1^−/−Lis1^+/− mutant was thinner at E12.5, but their CSPG positive zone was broadened (arrows). (C) At E13.5, while Nde1^+/−, Nde1^−/− and Nde1^+/−Lis1^+/− embryos all showed well separated preplate (indicated by yellow arrows), no splitting of the preplate could be detected in the Nde1^−/−Lis1^+/− cortex. In addition to greatly increased CSPG positive preplate neurons (in red), increases of DCX positive young cortical plate neurons (in green) was also detectable. E, Nde1; L, Lis1. Bar: 100 µm.

Figure 5.

Overproduction of C–R cells and enhanced Reelin signaling in the Nde1^−/−Lis1^+/− cortex. (A) Over production of Calretinin positive C–R cells (in red) was seen in the Nde1^−/−Lis1^+/− mutant. Significantly increased Calretinin positive cells were observed in the Nde1^−/−Lis1^+/− cortex at E12.5. E, Nde1; L, Lis1. Bar: 200 µm. (B and C) Over-production and abnormal positioning of Reelin secreting C–R cells in the Nde1^−/−Lis1^+/− mutant. Cortical sections were immunostained with two monoclonal antibodies to Reelin in red (CR50 and G10), and co-stained with an antibody to DCX in green and Hoechst in blue. The Nde1^−/−Lis1^+/− mutant showed dramatic increase and mis-localization of Reelin positive C–R cells throughout the entire course of corticogenesis starting from E11.5. Bar: 200 µm. (D) Immunoblotting analysis of Reelin protein levels in developing cerebral cortex. The cerebral cortices of E13.5 and E15.5 embryos were dissected and their total proteins extracts were analyzed on a 7.5% SDS–PAGE, followed by immunoblotting with an antibody to Reelin (G10). Loading is normalized by total protein amount and by immunoblotting with an antibody to tubulin. Bands on immunoblots were analyzed using Quantify One. Over 5-fold increase in Reelin protein (both 400 and 180 kDa bands) was detected in the Nde1^−/−Lis1^+/− mutant cortex over wild-type controls. (E) Down-regulation of Dab1 by increased Reelin signaling was observed in the Nde1^−/−Lis1^+/− mutant. Immunoblotting analysis was performed with protein extracts from the cortex of E15.5 embryos with antibodies to Dab1 and DCX. Compared to wild-type and Nde1^+/−Lis1^+/− mutant, a significant decrease in the Dab1 protein level was detected in the Nde1^−/−Lis1^+/−cortex, suggesting that the overproduced Reelin in the mutant were active and could elicited Dab1 degradation.

Figure 6.

Moderate changes in SVZ progenitor population by Nde1^−/−Lis1^+/− mutation. Double immunostaining of VZ progenitors with Pax6 (in red) and SVZ progenitors with Tbr2 (in green) at E12.5 (A) and E13.5 (B) demonstrated that the Nde1^−/−Lis1^+/− mutation lead to a relatively milder change in the number and density of SVZ progenitors than the VZ progenitors. Although the reduction of SVZ progenitor pool in the Nde1^−/−Lis1^+/− mutant became more detectable at E13.5, it is less significant compared to the dramatically diminished VZ progenitor pool. Numbers of VZ and SVZ cells per unit length of ventricular surface (Cell # per cortical column) were graphed. Ratios of SVZ/VZ progenitors at E12.5 and E13.5 were also presented. Bar: 100 µm.

Figure 7.

Mitotic defect in the Nde1^−/−Lis1^+/− mutant. (A) Abnormal mitotic orientation of Nde1^−/−Lis1^+/− cortical progenitors at E12.5. Anaphase progenitor cells were classified into three groups according to the angle of mitotic cleavage plane to the ventricular surface (vertical: 75–90°; horizontal: 0–25°; and diagonal: 25–75°). Approximately 300 anaphase cells were scored for each genotype. Percentage of each class of mitotic cleavage orientation from three litters with Nde1^+/−, Nde1^+/−Lis1^+/− and Nde1^−/−Lis1^+/− embryos were summarized. A moderate reduction in vertical cell cleavages and a corresponding increase in horizontal cell cleavages in the Nde1^−/−Lis1^+/−neural progenitors were detected (P < 0.01). Bar: 20 µm. (B) Phospho-Histone H3 (PH3) immunoreactivity demonstrated that a large fraction of mitotic progenitors failed to position metaphase chromosomes apically along the ventricular surface in Nde1^+/−Lis1^+/− and Nde1^−/−Lis1^+/− mutants at both E12.5 and E13.5 (P<0.001). However, the degree of abnormal mitotic nuclei positioning was equally severe in Nde1^+/−Lis1^+/− and Nde1^−/−Lis1^+/− mutants (P > 0.5). Bar: 100 µm.

Figure 8.

Morphology and cytoarchitectural defects of Nde1^−/−Lis1^+/ progenitors underlie the abnormal fate control. (A) Immunostaining the cell body of metaphase progenitors with a phosphorylated vimentin antibody 4A4 showed moderately reduced apical staining of VZ progenitors in the Nde1^−/−, Nde1^+/−Lis1^+/− mutants and a severe decrease in 4A4 immunoreactivity along the ventricular surface in the Nde1^−/−Lis1^+/− mutant (P<0.003), suggesting the destabilization and detachment of apical cell structures from the ventricular surface during metaphase. The abnormal 4A4 immunosignals in the VZ were indicated by arrows. Bar: 50 µm. (B) Electron micrograph of metaphase progenitors along ventricular surface, showing the reduced apical cell and membrane anchorage to ventricular surface, and weakened lateral cell–cell contacts of metaphase cells in the Nde1^−/−Lis1^+/−mutant. Red arrows indicate the electron dense adherens junctions formed between the apical end-feet of radial glial progenitors; green arrows indicate the membrane-like structures between the shrunken apical cell body. Shown are representative images of samples from three independent litters. Bars: 5 µm and 2 µm, respectively. (C) Immunohistological analysis of the apical membrane protein Prominin 1 showed that the space between the apical cell body of Nde1^−/−Lis1^+/− progenitors was occupied by the retracted apical membrane, further demonstrating the apical instability of the mutant progenitors. Bar: 50 µm. (D) Morphological and organizational abnormalities of Nde1^−/−Lis1^+/− progenitors were also viewed by labeling them with GFP through in utero electroporation. Although most of the Nde1^−/−Lis1^+/− progenitors showed well formed apical and basal end-feet, they frequently showed abnormal morphology of the cell body and processes. Two selected fields with different densities of GFP labeled progenitors were presented. Bar: 50 µm. (E) Abnormal metaphase cells with retracted thin apical processes and the formation of rosette-like mitotic cell clusters (indicated by asterisk) were also commonly seen in the Nde1^−/−Lis1^+/− mutant by co-immunolabeling with the radial glial marker GLAST. Bar: 20 µm. (F) Immunohistological analysis of radial glial marker GLAST and GFP labeling by in uteroelectroporation showed severe morphological defects of radial glial fibers near the MZ in the Nde1^−/−Lis1^+/− mutant, and suggested a role of Lis1–Nde1 in maintaining neural progenitor morphology at both apical and basal sides. Bars: 50 µm (GLAST) and 25 µm (GFP), respectively.

Figure 9.

A schematic presentation of cell fate control by the Lis1–Nde1 complex in radial glial progenitors, and its loss of functional defects. In normal metaphase radial glial progenitors, both the cell body and mitotic chromosomes are apically localized along the ventricular surface, and the cell is in close contact with neighboring progenitors. Such cellular architecture ensures the asymmetrical mitotic division and maintains the progenitor in proliferative state. In the Nde1^−/− and Nde1^+/−Lis1^+/− mutants, although many cells fail to position their mitotic chromosomes apically, large degree of apical cell–cell contacts still remains, resulting in moderate neurogenesis defects. Further loss of Lis1–Nde1 function in the Nde1^−/−Lis1^+/− progenitors results in destabilization of apical cell membrane and loss of cell–cell contacts. These changes in the cytoarchitecture of metaphase cells act collaboratively with the mitotic spindle orientation defects and induce striking shift of progenitor fate towards neuronal differentiation.