Fig. 3

NEUROD2 targets Cdk5r1 and Lrp8 genes during corticogenesis. a Diagrammatic representation of the Reelin signaling pathway that is required for cortical radial migration. Genes that are targets of NEUROD2 are marked with a red star. b NEUROD2 western blotting confirms knockdown in primary cortical neuronal culture transfected with Neurod2 shRNA as compared to a non-targeting shRNA. EGFP is used to verify transfection efficiency and BETA-ACTIN is used as a loading control. c, d and e mRNA expression data is obtained from Allen Developing Mouse Brain Atlas (http://developingmouse.brain-map.org) for Neurod2, Cdk5r1 and Lrp8 genes in E15.5 mouse brain. f and i NEUROD2 peaks associated with Cdk5r1 and Lrp8 are plotted relative to enhancer (H3K4me1) and promoter (H3K4me3) marks. H3K4me1 and H3K4me3 ChIP-Seq data is from (www.encodeproject.org) [31]. Peaks are plotted using the genome browser at genome.ucsc.edu. g and j NEUROD2 ChIP followed by qPCR confirms NEUROD2 binding to the promoter regions of Cdk5r1 and Lrp8 relative to a negative control (GFP ChIP). Data is normalized to the amount of input DNA as described in the Methods section (p-value < 0.05). Bars represent standard error of mean. Data represents two biological and five technical replicates. h and k Reverse transcription and qPCR analysis of neurons transfected with Neurod2 shRNA or non-targeting shRNA reveals a significant reduction in Cdk5r1 mRNA levels (p-value = 9.6x10⁻⁸) and a trend of reduction in Lrp8 levels (p-value = 0.18) after Neurod2 knockdown. All RT-qPCR results represented data from 3 biological samples each analyzed in technical triplicates. Bars represent standard error of mean