Quantitative real time analysis validates a transcriptional gene signature associated with ectoderm/epidermal development.

<p>qRT-PCR analysis of mRNA levels of genes associated with (A) growth factor genes (<i>FGF19</i>, <i>EGF</i>, <i>BMP10</i>), (B) transcriptional regulation (<i>NFATC1</i>, <i>DLX3</i>), (C) ectoderm development (<i>BARX2</i>, <i>FOXA2</i>, <i>LHX1</i>), (D) epithelial development (<i>IRF6</i>, <i>PAX6</i>) and (E) epidermal development (<i>TGM1</i>, <i>BNC1</i>) during the differentiation protocol (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122493#pone.0122493.g001" target="_blank">Fig 1A</a>) as compared to undifferentiated hESCs (n = 6 independent differentiation experiments for each bar). All data are ± SEM (*** p<0.001, ** 0.001</p

Analysis of in vivo expression and human diseases associated with genes upregulated in DAPT-treated hESCs during ectoderm specification.

<p>(A) Images of in situ localization of mRNAs expressed within the murine limb bud at E10.5-E11.5. Data are from the EMAGE gene expression database (EMAGE gene expression database (<a href="http://www.emouseatlas.org/emage/" target="_blank">http://www.emouseatlas.org/emage/</a>)), <i>P63</i> EMAGE:5000; <i>Cyp26b1</i> EMAGE:5746; <i>Capn</i> EMAGE:6170; <i>Vgll22</i> EMAGE:6040; <i>Fgf9</i> EMAGE:6189. Arrows indicate limb bud localization. (B) Analysis of mRNA expression of 211 upregulated genes with DAPT treatment of ectoderm specified hESCs was performed using EMAGE gene expression database (<a href="http://www.emouseatlas.org/emage/" target="_blank">http://www.emouseatlas.org/emage/</a>) and Gene Expression Database (<a href="http://www.informatics.jax.org/expression" target="_blank">http://www.informatics.jax.org/expression</a>). (C) Analysis of mRNA expression of 211 upregulated genes with DAPT treatment of ectoderm specified hESCs was performed using the human-mouse connection (<a href="http://www.informatics.jax.org/humanDisease.html" target="_blank">http://www.informatics.jax.org/humanDisease.html</a>). Diseases were categorized according to presentation of symptoms in indicated tissues.</p

DAPT treatment during ectoderm specification of hESC reveals a transcriptional gene signature associated with ectoderm/epidermal development.

<p>(A) Gene ontology analysis reveals a transcription gene signature associated with DAPT treatment during ectoderm specification of hESCs. (B) Gene ontology analysis reveals an upregulation of genes associated with ectoderm, epithelium and epidermal development with DAPT treatment during ectoderm specification of hESCs. The threshold of EASE Score, a modified Fisher Exact P-Value, for gene-enrichment analysis is depicted for specific annotation categories (p value ≤ 0.05 is considered strongly enriched). Genes highlighted in red were corroborated by quantitative real time PCR (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122493#pone.0122493.g004" target="_blank">Fig 4</a>).</p

DAPT accelerates ectoderm specification of hESCs.

<p>(A) Schematic of the protocol used to induce ectoderm specification of hESCs in the presence of an inhibitor of a <b>ϒ</b>-secretase inhibitor (DAPT) or vehicle control (ethanol). (B) Morphological analysis of phase images of hESCs colonies at different timepoints throughout the ectoderm specification protocol. DAPT-treatment accelerates colony differentiation as made evident by a qualitative increase in number of cells displaying a flattened morphological appearance. (C) Undifferentiated hESCs are negative for P63 (red) and AP2α (green), and are positive for the pluripotency markers SOX2 (red) and OCT4 (green). As cells differentiate, DAPT-treated colonies display an increase in P63 and AP2α expression and a loss in OCT4 and SOX2 at 3 and 6 days in differentiation conditions. Dotted line outlines colony edge and the asterisk marks undifferentiated areas of the colonies. (D) Quantitative real-time PCR (qRT-PCR) analysis of mRNA levels of <i>POU5F1</i> during differentiation as compared to undifferentiated hESCs (n = 6 independent differentiation experiments for each bar). (E) qRT-PCR analysis of mRNA levels of <i>TFAP2A</i> during differentiation as compared to undifferentiated hESCs (n = 3 independent differentiation experiments for each bar). All data are ± SEM (** 0.001</p

Quantitative analysis of differentially regulated genes in DAPT-treated hESCs during ectoderm specification.

<p>(A) Pie chart representation of the percentage of genes that are significantly upregulated and downregulated when hESCs are specified to surface-ectoderm cells in the presence of DAPT compared to vehicle-treated cells. (B) Heat map representation of differentially regulated genes in both the DAPT and vehicle-treated samples (in triplicate). Genes depicted in green correspond to genes that are significantly upregulated in the DAPT-treated sample versus the vehicle-treated samples (log2 fold change >1); genes depicted in red represent genes that are significantly downregulated (log2 fold change <1) in the DAPT-treated sample versus the vehicle-treated control.</p