Journal: Development (Cambridge, England)

Article Title: Mechanisms underlying WNT-mediated priming of human embryonic stem cells

doi: 10.1242/dev.200335

Figure Lengend Snippet: WNT priming and memory in hESCs. (A) Experimental conditions used to induce a WNT-primed or mesendodermal (ME) state. Cells were treated with 100 ng/ml Wnt3a (WNT) and 10 ng/ml activin A (ACT), either alone or sequentially, in E7 medium. (B) Expression of ME genes (BRA, EOMES and GSC) and pluripotent genes (NANOG, OCT4 and SOX2) measured by RT-PCR in cells treated with the experimental conditions defined in A. Data points represent the mean fold change relative to E7 for independent biological replicates. Bars and error bars represent the mean±s.d. across n=3 biological replicates. Differences between means that were determined to be significant are shown (*P<0.05, **P<0.01, ***P<0.001, ANOVA). (C) k-means clustering of RNA-seq data measured in four conditions (columns) revealed four clusters that could be annotated as follows: genes for which the expression increases in all conditions in which activin is present (cluster 1, n=349); genes for which the expression increases in all conditions in which WNT is present (cluster 2, n=206); genes that are further activated by WNT and activin (higher induction in WNT/ACT than WNT or ACT alone, cluster 3, n=222); or genes that are repressed by the WNT/ACT condition (cluster 4, n=435). The intensities represent log2 fold change of expression relative to the E7 condition (color is shown in the scale bar). Only genes showing a significant change (adjusted P-value<0.01) in at least one condition relative to E7 were included in the heatmap and clustering procedure. (D) The memory of the WNT-primed state was tested by incubating cells for an additional day in E7 base medium in the presence or absence of SB431542 (SB; 10 μM) prior to presenting activin (10 ng/ml). SB was used to eliminate activin signaling during the first 48 h. After 24 h of activin stimulation, cells were fixed and analyzed by immunofluorescence (IF) for BRA and SOX2 expression. Scale bar: 50 µm. (E) Violin plots of the nuclear IF signal quantified in single cells (n>5000 cells per condition) from the experiment shown in D.

Article Snippet: The following primary antibodies and dilutions were used: SOX2 (rabbit monoclonal, Cell Signaling Technology, 3579, 1:200), brachyury (goat polyclonal, R&D Systems, AF2085, 1:150), brachyury (rabbit monoclonal, R&D Systems, MAB20851, 1:400), EOMES (mouse monoclonal, R&D Systems, MAB6166, 1:400), GSC (goat polyclonal, R&D Systems, AF4086, 1:200).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, RNA Sequencing, Immunofluorescence

Journal: Development (Cambridge, England)

Article Title: Mechanisms underlying WNT-mediated priming of human embryonic stem cells

doi: 10.1242/dev.200335

Figure Lengend Snippet: Presence of nuclear β-catenin is not sufficient for ME differentiation. (A) Schematic depicting the hypothesis to be tested that increased ME gene expression requires overlap of β-catenin and SMAD2/3. Cartoon showing the nuclear concentration of β-catenin and SMAD2 during the WNT/ACT treatment. (B) Method for testing the hypothesis in A using shorter WNT treatment. IWP2, which prevents cells from secreting WNT ligands, is used in this protocol to ensure that WNT signaling is restricted to the first 6 h. (C) Short WNT exposure is not sufficient to prime the cells for ME differentiation. Cells were treated for different amounts of time with WNT (100 ng/ml) prior to adding activin (ACT; 10 ng/ml). After 24 h of activin stimulation, cells were fixed and analyzed by immunofluorescence for EOMES, BRA and GSC expression. Violin plots of the nuclear IF signal quantified in single cells (n>10,000 cells per condition). Solid line, median; dashed lines, upper and lower quartiles. (D) Example images corresponding to the analysis shown in C. Scale bar: 50 µm.

Article Snippet: The following primary antibodies and dilutions were used: SOX2 (rabbit monoclonal, Cell Signaling Technology, 3579, 1:200), brachyury (goat polyclonal, R&D Systems, AF2085, 1:150), brachyury (rabbit monoclonal, R&D Systems, MAB20851, 1:400), EOMES (mouse monoclonal, R&D Systems, MAB6166, 1:400), GSC (goat polyclonal, R&D Systems, AF4086, 1:200).

Techniques: Gene Expression, Concentration Assay, Immunofluorescence, Expressing

Journal: Development (Cambridge, England)

Article Title: Mechanisms underlying WNT-mediated priming of human embryonic stem cells

doi: 10.1242/dev.200335

Figure Lengend Snippet: The EOMES motif is enriched in WNT/ACT-enhanced ATAC peaks. (A) Schematic depicting the hypothesis to be tested that increased mesendodermal (ME) gene expression requires a SMAD2/3 co-activator (X) that is induced by WNT. (B) Heatmap of the ATAC-seq signals that are enhanced in the WNT/ACT condition in comparison with WNT or ACT alone. (C) Probability of finding these WNT/ACT-enhanced peaks near genes (start of the gene −20 kb to end of the gene +20 kb) that are either sensitive or insensitive to WNT priming. ***P<0.001. (D) The EOMES motif is the most enriched motif, followed by brachyury (BRA), which is found in the WNT/ACT-enhanced peaks near genes that are WNT primed.

Article Snippet: The following primary antibodies and dilutions were used: SOX2 (rabbit monoclonal, Cell Signaling Technology, 3579, 1:200), brachyury (goat polyclonal, R&D Systems, AF2085, 1:150), brachyury (rabbit monoclonal, R&D Systems, MAB20851, 1:400), EOMES (mouse monoclonal, R&D Systems, MAB6166, 1:400), GSC (goat polyclonal, R&D Systems, AF4086, 1:200).

Techniques: Gene Expression, Comparison