Three-Dimensional Regulation of Radial Glial Functions by Lis1-Nde1 and Dystrophin Glycoprotein Complexes

Lis1-Nde1 integrates cerebral cortical neurogenesis with neuronal migration by stabilizing the basal-lateral surface of radial glial cells

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

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

The Lis1–Nde1 complex is essential for determining cerebral cortical size, shape and lamina structures

() Dying at birth, the brains of Nde1Lis1 mutant mice were dramatically reduced (arrows). A more pronounced size reduction of the cerebral hemispheres was observed. () Histological analysis showed that the neocortex of the Nde1Lis1 mutant was severely disorganized, lacked the normal MZ and any other cortical layers. () Immunohistological analyses with layer-specific markers showed that in the Nde1Lis1 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 () Rostral to caudal length (L1), medial to lateral length (L2) and cortical thickness (V) of Nde1Lis1 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).<p><b>Copyright information:</b></p><p>Taken from "Lis1–Nde1-dependent neuronal fate control determines cerebral cortical size and lamination"</p><p></p><p>Human Molecular Genetics 2008;17(16):2441-2455.</p><p>Published online 10 May 2008</p><p>PMCID:PMC2486443.</p><p>© 2008 The Author(s).</p

Overproduction of C–R cells and enhanced Reelin signaling in the Nde1Lis1 cortex

() Over production of Calretinin positive C–R cells (in red) was seen in the Nde1Lis1 mutant. Significantly increased Calretinin positive cells were observed in the Nde1Lis1 cortex at E12.5. E, Nde1; L, Lis1. Bar: 200 µm. ( and ) Over-production and abnormal positioning of Reelin secreting C–R cells in the Nde1Lis1 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 Nde1Lis1 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. () 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 Nde1Lis1 mutant cortex over wild-type controls. () Down-regulation of Dab1 by increased Reelin signaling was observed in the Nde1Lis1 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 Nde1Lis1 mutant, a significant decrease in the Dab1 protein level was detected in the Nde1Lis1cortex, suggesting that the overproduced Reelin in the mutant were active and could elicited Dab1 degradation.<p><b>Copyright information:</b></p><p>Taken from "Lis1–Nde1-dependent neuronal fate control determines cerebral cortical size and lamination"</p><p></p><p>Human Molecular Genetics 2008;17(16):2441-2455.</p><p>Published online 10 May 2008</p><p>PMCID:PMC2486443.</p><p>© 2008 The Author(s).</p

Overproduction of PP neurons and lack of preplate splitting in the Nde1 Lis1 cortex

() 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 Nde1Lis1 cortex before its size was reduced at E11.5. () The cortex of Nde1Lis1 mutant was thinner at E12.5, but their CSPG positive zone was broadened (arrows). () At E13.5, while Nde1, Nde1 and Nde1Lis1 embryos all showed well separated preplate (indicated by yellow arrows), no splitting of the preplate could be detected in the Nde1Lis1 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.<p><b>Copyright information:</b></p><p>Taken from "Lis1–Nde1-dependent neuronal fate control determines cerebral cortical size and lamination"</p><p></p><p>Human Molecular Genetics 2008;17(16):2441-2455.</p><p>Published online 10 May 2008</p><p>PMCID:PMC2486443.</p><p>© 2008 The Author(s).</p

Reduction of VZ progenitors at the onset of corticogenesis by Lis1–Nde1 double deficiency

() The size of telencephalic vesicles (indicated by dashed blue circles) of the Nde1Lis1 mutant appeared close to normal before E11, but was significantly smaller by E13 compared to that of their littermates. () 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 Nde1Lis1 mutant. < 0.001. E, Nde1; L, Lis1. Bar: 100 µm. () Reduced neural progenitors in the Nde1Lis1 mutant cortex was indicated by reduced immunostaining of the glutamate transporter GLAST (in red), a marker of radial glial progenitors. Bar: 100 µm. () Substantial cell death was detected in the cerebral cortex of the Nde1Lis1 mutant by TUNEL staining (in green). In contrast, very few TUNEL positive cells were detected in the Nde1 and Nde1Lis1 controls. Bar: 100 µm. () Majority (over 80%) of TUNEL positive cells (in green) in the Nde1Lis1 mutant were newborn post mitotic neurons in the intermediate zone (IZ) and the cortical plate (CP) and expressed DCX (in red). Bar: 100 µm. () Cell-cycle exit profiles of Nde1Lis1progenitors. 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 Nde1Lis1 cortex by E13, while only 25–28% of the Nde1Lis1 or Lis1 progenitors left the cell cycle (<p><b>Copyright information:</b></p><p>Taken from "Lis1–Nde1-dependent neuronal fate control determines cerebral cortical size and lamination"</p><p></p><p>Human Molecular Genetics 2008;17(16):2441-2455.</p><p>Published online 10 May 2008</p><p>PMCID:PMC2486443.</p><p>© 2008 The Author(s).</p

Morphology and cytoarchitectural defects of Nde1Lis1 progenitors underlie the abnormal fate control

() Immunostaining the cell body of metaphase progenitors with a phosphorylated vimentin antibody 4A4 showed moderately reduced apical staining of VZ progenitors in the Nde1, Nde1Lis1 mutants and a severe decrease in 4A4 immunoreactivity along the ventricular surface in the Nde1Lis1 mutant (<p><b>Copyright information:</b></p><p>Taken from "Lis1–Nde1-dependent neuronal fate control determines cerebral cortical size and lamination"</p><p></p><p>Human Molecular Genetics 2008;17(16):2441-2455.</p><p>Published online 10 May 2008</p><p>PMCID:PMC2486443.</p><p>© 2008 The Author(s).</p