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. 2008 May 10;17(16):2441–2455. doi: 10.1093/hmg/ddn144

Figure 5.

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.