Fig. 5

NEUROD2 target genes encode transcription factors that control the differentiation of distinct populations of cortical layer specific projection neurons. mRNA expression data is obtained from Allen Developing Mouse Brain Atlas (http://developingmouse.brain-map.org) for Fezf2 (a), Bcl11b (e), Cux1 (i) and Satb2 (m) for E15.5 mouse brain. Neurod2 expression overlaps with the domains expressing each of the cortical layer specific transcription factors. (b, f, j and n) NEUROD2 peaks 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. (c, g, k and o) NEUROD2 ChIP followed by qPCR confirms NEUROD2 binding to the promoter regions of all four layer markers relative to a negative control (GFP ChIP) (p-value < 0.05). Data is normalized to the amount of input DNA as described in the Methods section. Bars represent standard error of mean. Data represents two biological and five technical replicates. (d, h, l and p) mRNA levels of Fezf2, Bcl11b, Cux1 and Satb2 were quantified by RT-qPCR in neurons transfected with either a non-targeting or a Neurod2 shRNA. A significant reduction in Cux1 mRNA levels was observed in the absence of NEUROD2. Each experiment represents 3 biological samples each analyzed in technical triplicates. Data is presented as fold change of control sample (* p-value = 0.0014). p-values for changes in expression of Fezf2, Bcl11b and Satb2 were between 0.1 and 0.2. A non-targeting shRNA was used as control. Bars are standard error of the mean