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. 2021 Aug 26;12:5140. doi: 10.1038/s41467-021-25237-8

Fig. 3. Roles of WNT, TGF-β, and BMP pathways in T/Bra expression dynamics.

Fig. 3

a, b Representative images showing T/Bra:mCherry (top), TLC:mCherry (middle), and AR8:mCherry (bottom) expression dynamics in epithelialized (a) and non-epithelialized (b) EpiTS embryoids between 72 and 120 h. c–e Background normalized mean intensity of T/Bra:mCherry (c), TLC:mCherry (d), and AR8:mCherry (e) in epithelialized or non-epithelialized embryoids between 72 and 120 h. Data were collected from three biologically independent experiments. For d, adjusted p-values are: Non-epith.+TS (72 h) vs Non-epith.+TS (96 h), p = 0.0168. f Representative images at 120 h showing T/Bra:mCherry expression in epithelialized embryoids treated with indicated inhibitors between 96 and 120 h. g Percentage of T/Bra:mCherry-positive epithelialized embryoids at 120 h, treated with indicated inhibitors between 72 and 96 h or 96 and 120 h. Background offset was used to set a threshold for T/Bra:mCherry expression. Adjusted p-values are: Control vs Lefty (96–120 h), p = 0.0061; Control vs Lefty (72–96 h), p = 0.0003; Control vs Noggin (72–96 h), p = 0.0033; Noggin (96–120 h) vs Dkk1 (96–120 h), p = 0.0001; Noggin (96–120 h) vs Lefty (72–96 h), p = 0.0121; Noggin (96–120 h) vs Dkk1 (96–120 h), p = 0.0001; Noggin (96–120 h) vs Dkk1 (72–96 h), p = 0.0006; Dkk1 (96–120 h) vs Lefty (96–120 h), p = 0.0325. h Timelapse analysis between 78 and 192 h with 2 h interval showing the onset of T/Bra expression. For control, Dkk1, Lefty, Noggin conditions, total number of embryoids analyzed were 23, 18, 15, 20, respectively. Data are collected from a single experiment. Adjusted p-values are: Control vs Dkk1, p = 0.0002; Noggin vs Dkk1, p = 0.0007. i, j Quantification of coverage index of T/Bra:mCherry expression (i) and EPI compartment area (j) at 120 h in epithelialized embryoids treated with indicated inhibitors. For i, adjusted p-values are: Control vs Dkk1 (72–96 h), p = 0.0123; Control vs Lefty (72–96 h), p = 0.0010; Control vs Lefty (96–120 h), p = 0.0009; Control vs Noggin (96–120 h), p = 0.0300. For j, adjusted p-values are: Control vs Lefty (72–96 h), p = 0.0276; Dkk1 (72–96 h) vs Lefty (96–120 h), p = 0.0049; Noggin (72–96 h) vs Lefty (96–120 h), p = 0.0021. k Representative images showing T/Bra:mCherry expression in epithelialized embryoids treated with indicated small molecule inhibitors between 96 and 120 h. l Percentage of T/Bra:mCherry-positive epithelialized embryoids at 120 h, treated with indicated inhibitors between 96 and 120 h. All embryoids were formed from 100ESC/100TSC condition. For all conditions in a–e, f, g, i, j, and k, l number of mCherry-positive embryoids over total number of embryoids analyzed are indicated at bottom right of a–b, f, and k, respectively. Large symbols indicate mean values of each replicate. Black lines indicate median and quartiles. For all statistical analysis, one-way ANOVA followed by Tukey multiple comparison test was performed. For g, i, and j significance was calculated in comparison to control. Following P value style was used: (****) < 0.0001, (***) 0.0002, (**) 0.0021, (*) 0.0332, (ns) 0.1234. Scale bars: 200 µm. Source data are provided as a Source Data file.