Alternative early neural differentiation to neural crest progenitors.

<p>(A) hESC were differentiated towards NCP and stained with antibodies specific for <i>OCT4</i> (no stain observed), <i>PAX6</i> (no stain observed), <i>NESTIN</i> and <i>HNK-1</i>. Cell nuclei were labeled with the DNA dye Hoechst H-33342 (blue). Scale bars: 100 µm. (B) Pairwise comparisons of hESC, NEP or NCP yielded 4277 differentially expressed transcripts. The heat map displays the genes after clustering according to the Pearson's correlation of their expression values across samples. The colors represent Z-scores of the row-wise normalized expression values for each gene. The dendrogram indicates the pattern similarities indicated by Spearman correlation distances (1- Spearman correlation coefficient) and shows a large separation of NCP from NEP and hESC. (C) The expression of early neuronal marker genes was measured in three preparations each of hESC, NEP and NCP by qPCR. The transcript levels of NEP and NCP were calculated relative to hESC. The relative gene expression levels were color coded (significant down-regulation vs. hESC in blue; significant up-regulation in red; non-significant changes marked by “N”. The genes showing different behavior in NEP vs NCP are displayed. All measures of variance and p-values are indicated in the supplemental material.</p

Transcriptional regulation of epigenetic modifiers during neuroepithelial differentiation.

<p>(A) The levels of epigenetic modifier transcripts of NEP and hESC were analyzed by qPCR in three independent cell preparations, and relative abundances were calculated. The data were color-coded, with up-regulated genes displayed in red and down-regulated genes in blue. Measures of variance and p-values are indicated in supplemental material, and only significantly regulated genes are displayed. Genes up-regulated >5-fold are displayed in bold. (B) The transcript levels of HDACs were determined for hESC, NEP, NCP and CTX. All expression levels of differentiated cells were normalized to those of hESC, and relative abundances are displayed. For instance, seven different HDACs were up-regulated in NEP compared to hESC. The dotted lines indicate 2-fold regulation levels. Data are means ± SEM of three independent differentiations. *: p<0.05</p

Effect of neuroectodermal differentiation on localization of histone marks.

<p>(A) hESC were differentiated towards NEP and stained with antibodies specific for Oct4, HNK-1 (neural crest marker), Pax6 (NEP marker) and nestin (neural stem cell marker). Nuclei were stained with the DNA dye H-33342 (blue). Scale bars: 100 µm. (B) GO analysis of the up-regulated genes in NEP compared to hESC (C) Whole cell extracts from hESC and NEP were analyzed by Western blot with antibodies specific for the indicated histone H3 modifications. Total histone H3 (Pan-H3) was used as loading control. (D) hESC and differentiated NEP were grown on glass cover slips and immunostained with antibodies specific for H3K9me3 or H4K20me3. The upper panels show grey-scale signal intensities of the stain, the lower panels show a superimposition of the same histone stain as above (red) with a DNA counter-stain (DAPI, blue). Arrows mark two cells with a diffuse H4K20me3 stain, which differs from the spot-like pattern always observed in hESC. Scale bars: 10 µm. (E) Chromatin immunoprecipitiation was perfomed from nuclei of hESC or NEP with antibodies specific for H3K4me3 and H3K27me3. The abundance of promoter regions of <i>OCT4, NANOG, PAX6</i> and <i>SOX2</i> was measured by qPCR with specific primers for the indicated genes. Data were compared to control samples prepared without specific antibody and are indicated as relative enrichment. Data are means ± SD from 2 experiments.</p

Compilation of 150 representative genes involved in epigenetic regulation.

<p>The genes were identified, selected, and classified according to their function based on published literature and database searches. In a further step, the genes related to histone modifications were sub-classified as writers (coding for modifying enzymes such as methyltransferases), as erasers (coding for de-modifying enzymes such as histone deacetylases) and as readers (coding for proteins that bind to the respective modification).</p

Regulation of epigenetic modifiers in NCP, and their comparison to NEP.

<p>The levels of epigenetic regulator gene transcripts were measured in hESC, NCP and NEP by qPCR, and the expression levels were calculated relative to the levels in hESC. All genes that showed significant up- or downregulation in NCP compared to hESC are displayed. The relative expression level (vs. hESC) was color coded, as illustrated in the chromatic scale in the bottom. Genes with >5-fold expression in NCP vs. hESC are shown in bold. For better comparison, the data for the same genes are shown for NEP in the right-hand column. Measures of variance and p-values are indicated in the supplemental material.</p

Synopsis of the regulation of different epigenetic modifier groups at different stages of neuronal differentiation.

<p>Four groups of epigenetic modifiers were selected for a comparison of relative expression levels of NEP, NCP and CTX. Data were obtained, and significances calculated as described earlier. All data are means ± SEM of three independent differentiations. (A) Genes that code for subunits of the BAF remodeling complex. (B) Genes that code for histone acetyl transferases (HAT). (C, D) Genes that are involved in PRC1 and PRC2 complex formation. *: p<0.05 vs hESC transcript level.</p