5 suggested that Robo signaling might influence neurogenesis in a more direct manner. To test this hypothesis, we examined the status of three of the main signaling pathways controlling cortical neurogenesis, Notch, fibroblast growth factor (FGF), and WNT, by analyzing the expression of their effector

genes Hes1, Spry2, and Axin2, respectively. buy NVP-BKM120 Using quantitative PCR (qPCR), we found that the expression of basic helix-loop-helix (bHLH) gene Hes1 was significantly reduced in the cortex of E12.5 Robo1/2 mutants compared to controls ( Figure 7A). In contrast, no significant changes were observed in mRNA levels for Spry2 and Axin2 ( Figure S8A). Thus loss of Robo signaling seems to disrupt the expression of the Notch signaling effector

Hes1 in the absence of generalized changes in other important signaling pathways that are known to be active in progenitor cells. We next examined the expression of several other components of the Notch signaling pathway. We found no significant changes in total mRNA levels for the Notch ligand Dll1, Notch1, or Hes5, another target gene of Notch signaling ( Figure 7A; data not shown). mRNA analysis by in situ hybridization Ku-0059436 solubility dmso confirmed the reduction of Hes1 in progenitor cells of the cerebral cortex ( Figures 7B and S8B). In addition, it revealed that expression of Dll1, which is negatively regulated by Hes1, was increased in scattered cells throughout the VZ of the Robo1/2 mutant cortex compared to controls ( Figures 7B and S8B). A reduction in Hes1

levels could explain the decreased number of VZ mitosis and the increase in IPCs found in the Robo1/2 mutant cortex, because Hes1 unless expression is thought to maintain the status of progenitor cells in the VZ ( Ishibashi et al., 1994; Nakamura et al., 2000). To experimentally test this hypothesis, we first attempted to rescue the IPC phenotype observed in Robo1/2 mutants by overexpressing Hes1. To this end, we electroporated a plasmid encoding Gfp, alone or in combination with full length Hes1, in the cortex of Robo1/2 mutant embryos at E12.5 and analyzed the expression of Tbr2 in electroporated cells 24 hr later ( Figure 7C). We found that overexpression of Hes1 in Robo1/2 mutant progenitor cells dramatically reduced the fraction of Tbr2+ cells within the electroporated clones ( Figures 7D–7F). In reciprocal experiments, we knocked down Hes1 protein levels by using RNA interference. In brief, we electroporated chemically synthesized small interference RNA (siRNA) that has been previously shown to produce significant knockdown of mouse Hes1 ( Noda et al., 2011; Ross et al., 2004) or control siRNA, along with a plasmid encoding Gfp, in the cortex of wild-type embryos at E12.5 and analyzed the expression of Tbr2 in electroporated cells 48 hr later ( Figure 7G).