These experiments suggest the DISC1 A83V, R264Q, and L607F variants are not able to function similarly to WT-DISC1 in the regulation of neural progenitor proliferation. We then directly addressed if the changes

in BrdU labeling were due to alterations in the numbers of progenitors exiting the cell cycle and prematurely differentiation. First, we performed the cell cycle exit assay in utero whereby electroporated brain sections were stained for GFP, BrdU, and Ki67. To assess the cell cycle exit index, we counted the percentage of GFP/BrdU double-positive cells that were negative for Ki67, Using this protocol, we found that overexpression of WT-DISC1 was able to rescue the increased cell cycle exit mediated by DISC1 shRNA (Figure 3B). Upon comparison to the DISC1 variants, we found that the A83V, R264, and L607F check details variants all were not able to rescue similar to WT-DISC1 and neural progenitor cells continued to prematurely exit the cell cycle.

However, the S704C variant was able to rescue the DISC1-shRNA-mediated increase in cell cycle exit (Figure 3B), in good agreement with the neural progenitor proliferation data in Figure 3A. We extended these experiments to determine whether the changes in cell cycle exit led to alterations in neuronal differentiation. Electroporated brain sections were costained with GFP and Tuj1 to visualize neurons. We determined that selleckchem the increase in the number

of double-positive GFP/Tuj1 cells due to DISC1 shRNA was rescued when coexpressed with human WT-DISC1 (Figure 3C). In this assay, we observed that the A83V, R264Q, and L607F variants all did not increase the number of double-positive GFP/Tuj1 cells, while the S704C variant indeed functioned similar to WT-DISC1 and rescued similarly (Figure 3C). Together these data suggest that the A83V, R264Q, and L607F DISC1 variants do not function similar to WT-DISC1 or S704C in the regulation of neural progenitor proliferation. To determine whether the 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase DISC1 variants possessed dominant-negative activity in the presence of endogenous mouse DISC1, we performed the in utero electroporation and only overexpressed GFP, WT-DISC1, or the different variants. Staining for BrdU and GFP revealed that overexpression of human WT-DISC1 resulted in a significant increase in the percentage of cells double positive for GFP and BrdU demonstrating that WT-DISC1 expression alone increases the number of dividing neural progenitor cells (Figure S2A). Comparison to the DISC1 variants revealed that the S704C variant similarly increased the number of cycling cells as WT-DISC1. However, the A83V and L607F variant conditions revealed statistically similar numbers of GFP/BrdU-positive cells as GFP controls.