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. Author manuscript; available in PMC: 2017 Jul 20.
Published in final edited form as: In Vitro Cell Dev Biol Anim. 2016 Jun 1;52(9):961–973. doi: 10.1007/s11626-016-0056-7

Figure 3.

Figure 3

Role of Ascl1, Smad7, and Nr2f1 induction in the emergence of neural transcriptome in differentiating ESCs. (A) Heatmap for expression of selected neural genes in cells with induced Ascl1 (Dox−) and control cells (Dox+) on days 3, 5, 7, 10, and 14 in culture (see conditions in Fig. 1B); baseline—gene expression in ESCs day 3 in Dox+ medium; target genes of Ascl1 are marked +++ if they had >2 binding sites within 100 kb from transcription start site (TSS) and at least one was supported by >2 data sources, ++ if they had two binding sites and at least one was supported by >2 sources, and + if they had binding sites supported by only two sources. (B) Parametric analysis of gene set enrichment (PAGE) of target genes of Ascl1 (binding sites supported by >2 data sources with 100 kb from TSS) among genes that were upregulated in cells with induced Ascl1at time points 3, 5, 7, 10, and 14 d; baseline—expression in control cells (Dox+) at the same time point. (C) Rank-plot for Ascl1 target genes (same as in (B)) in cells with induced Ascl1 on day 5; horizontal axis represents genes sorted by expression change in Dox− versus Dox+, and the graph shows the proportion of Ascl1 target genes in a sliding window of 300 genes. (D) Heatmap for expression of miRNA that were significantly upregulated in cells with induced Ascl1 (Dox−) versus control cells (Dox+) on days 3, 5, 7, 10, and 14 in culture (see conditions in Fig. 1B); baseline—same as in (A); target genes of Ascl1 are marked +++, ++, or + as in (A). (E) Correlation of gene expression profiles in neuron-like cells derived from ESCs by the induction of TFs and in various mouse tissues and cultured cells; blue bars—cells enriched by microbeads, and orange bars—remaining cells after enrichment; gene expression values were log-transformed and normalized by maximum expression of each gene in ESCs on day 3 in Dox+ and median expression in non-neural tissues. (F) Number of neural-related genes upregulated by ≥2-fold in PSA-NCAM(+) cells generated with Ascl1, Smad7, and Nr2f1 and in various mouse tissues and cultured cells; baseline—maximum expression of each gene in ESCs on day 3 in Dox+ and median expression in non-neural tissues.