Extended Data Fig. 3: Differential gene expression during cell sorting and perturbations.
From: Reconstruction and deconstruction of human somitogenesis in vitro
a, Expression fold change plots of selected adhesion proteins between MESP2-low vs MESP2-high cells at 72 h, or between MESP2-high vs UNCX cells at 120 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n). The genes plotted are differentially expressed cadherin and protocadherin encoding genes from the comparison between MESP2-low and MESP2-high cells at 72 h. The dashed (dark red) lines represent log2 fold change values of −0.58 and 0.58. The error bar represents the estimated standard error for the log fold change from the model (DESeq2) which is represented as the centre of the bar. Genes with fold changes greater than 1.5 (above or below the dash line) and padj < 0.05 (estimated by Deseq2 using two-sided Wald test) are considered to be differentially expressed and coloured in either yellow (upregulated in MESP2 low cells) or magenta (upregulated in MESP2 high cells). Genes in blue colour from the comparison between MESP2-high and UNCX cells at 120 h are non-differentially expressed genes. The exact P values for each gene are shown in Supplementary Table 3. b, Normalized RNA counts of selected genes encoding adhesion proteins in MESP2-high and MESP2-low cell fractions at 72 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n; DESeq2 with two-sided Wald test). Cells with top 10% mCherry fluorescence are shown on the left (magenta) and top 10% YFP fluorescence on the right (yellow). c, Expression fold change plots of selected Ephrin protein encoding genes between MESP2-low vs MESP2-high cells at 72 h, or between MESP2-high vs UNCX cells at 120 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n). The error bar represents the estimated standard error for the log fold change from the model (DESeq2) which is represented as the centre of the bar. Genes with fold changes greater than 1.5 (above or below the dash line) and padj < 0.05 (estimated by Deseq2 using two-sided Wald test) are considered to be differentially expressed and coloured in either yellow (upregulated in MESP2 low cells) or magenta (upregulated in MESP2 high cells). Genes in blue colour from the comparison between MESP2-high and UNCX cells at 120 h are non-differentially expressed genes. The exact P values for each gene are shown in Supplementary Table 3. d, Normalized RNA counts of selected genes encoding Ephrin proteins in MESP2-high and MESP2-low cell fractions at 72 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n; DESeq2 with two-sided Wald test). e, Expression fold change plots of selected cytoskeleton regulating proteins between MESP2-low vs MESP2-high cells at 72 h, or between MESP2-high vs UNCX cells at 120 h (n = 3 independent experiments for each time point, with 96 Somitoids in each n). The error bar represents the estimated standard error for the log fold change from the model (DESeq2) which is represented as the centre of the bar. Genes with fold changes greater than 1.5 (above or below the dash line) and padj < 0.05 (estimated by Deseq2 using two-sided Wald test) are considered to be differentially expressed and coloured in either yellow (upregulated in MESP2 low cells) or magenta (upregulated in MESP2 high cells). Genes in blue colour from the comparison between MESP2-high and UNCX cells at 120 h are non-differentially expressed genes. The exact P values for each gene are shown in Supplementary Table 3. After differential gene expression analysis using Deseq2, differentially expressed genes from the MESP2-high vs MESP2-low comparison (72 h) were used to do KEGG functional analysis. The 42 genes plotted represent those that appear in the KEGG pathway “hsa04810” (Regulation of actin cytoskeleton). f, Kymograph of HES7 and MESP2 reporters obtained from a line scan across the centre of a Somitoid overexpressing Tiam1 induced by Doxycycline addition at 48 h. g, percentage of UNCX-positive cells characterized by flow cytometry in 120 h control and Somitoids overexpressing Tiam1 induced by Doxycycline addition at 48 h. Bars represent median. Unpaired two-tailed t-test n = 6 replica from 2 independent experiments, with 12—18 Somitoids in each replica. h, Left, maximum-z-projection confocal image of a MESP2/UNCX-reporting Somitoid at 120 h, overexpressing Tiam1 induced by Doxycycline addition at 48 h. Right, spatial auto-correlation analysis of MESP2 and UNCX signals (n = 3 Somitoids for each condition). All scale bars represent 500 µm.
