Fig. 1

Scl, Mesp1 and Tbx6 are transiently co-expressed in single cells. a Top, Schematic of ES/EB in vitro differentiation. Right and bottom, RT-qPCR gene expression analyses from RNA isolated from day 2–6 EB cells (n = 3, mean ± SD). b Example of detection of single mRNA molecules (foci) in a single EB cell by smRNA FISH. Left, raw image. Right, foci detection (green squares) with ImageJ Macro. c Average number of mRNA molecules/cell determined by smRNA FISH for Scl, Mesp1 and Tbx6, from day 3 to 4.5 EBs. Cell no.: number of cells analysed at each time point. d Distribution of Scl-positive cells (Scl/Mesp1/Tbx6, Scl/Tbx6, Scl/Mesp1, Scl-only) in day 3, 3.5, 4, and 4.5 EBs. Total number of EB cells and percentage of Scl-positive cells are indicated for each timepoint. Cells are considered positive for a marker when harbouring 6 or more foci. e smRNA FISH images showing a negative cell (top panel) and a cell positive for Scl, Mesp1 and Tbx6 (bottom panel) from day 3.5 EBs. Arrows indicate typical foci for each mRNA species; white star, background signal. f Significant non-linear negative correlation of expression between Scl and Mesp1 and Scl and Tbx6. Each dot represents a cell from day 3.5 EBs (total cells: 734). X-axis, number of Scl foci per cell; Y-axis, Mesp1 foci; Z-axisTbx6 foci. Numbers of Tbx6 foci are also indicated by a grey-red scale. Examples of Scl/Mesp1 and Scl/Tbx6 negatively correlated cells (i, ii, iii; Fig. 1g) are marked. Correlation coefficients: Scl/Mesp1: −0.2379, p-value < 0.0001, 95% confidence interval (CI): −0.3117/−0.1612; Scl/Tbx6: −0.1504, p-value < 0.002, 95% CI −0.2274/−0.07151. N, L, H: number of Scl foci/cell (N, negative; L, low (6–20 foci); H, high (21–139 foci)). g smRNA FISH images of representative cells showing Scl^high/Mesp1^low (i, left) and Scl^high/Tbx6^low (i, right), Scl^low/Mesp1^high (ii), Scl^low/Tbx6^high (iii) mRNA foci. Scale bars: 11.3 μm. See also Supplementary Fig. 2